This post is the third in a series (Subtle Environmental Poisons and Disease) dealing with environmental toxic influences, particularly those with long-term ‘subtle’ action. The major subtheme here is the role of individual variation in determining the outcome of a toxic challenge, with particular emphasis on how (in some cases) an organism’s anti-toxic protective mechanism may actually be a source of problems. An implicit requirement underlying both of these topics is the existence of specialized systems for recognizing potentially dangerous non-self molecules from the environment. These themes accordingly center around xenorecognition, or the ability to recognize foreign chemical intrusion at the molecular level. Framing the matter in this manner may bring to mind the immune system, and indeed an analogy can be made between responses to chemical intrusion and innate immune systems tuned by evolution for signaling responses to the presence of dangerous pathogenic organisms. Although such parallels should not be overstated, both systems serve to maintain homeostasis for complex multi-cellular organisms.
Contending with a Sea of Potential Poisons
The exquisite complexity of living biosystems dictates their sensitivity to a variety of negative perturbations, which can range across a spectrum of extraneous physical, chemical and biological influences. Parasitic replicating systems are likely to have been a serious problem even at the earliest stages of molecular evolution, and defenses against them likewise must have evolved at equally early times. It is precisely the ability of invasive biosystems to replicate at a host organism’s expense which renders such parasites a serious threat. When replication per se is combined with the frequent ability of biological invaders to rapidly evolve (and alter the sets of nucleic acids and proteins by which they may be recognized), a potent selective force is generated for the evolutionary derivation of increasingly complex counter-attacks. These we refer to as immune systems.
Yet there are a great many environmental potential threats which do not directly replicate, and these may originate from both biological or non-living sources. For the latter, we could think of toxic levels of metal ions or other soluble inorganic natural chemicals (such as dangerous oxygen radicals), or natural sources of dangerous gases (such as from volcanic effluxes). Across the field of biology in general, there is a huge range of natural poisons enzymatically synthesized from bacterial, fungal, plant or animal sources. As enzyme systems evolve, so too their range of natural products will change. Given these factors, the sheer numbers of potentially toxic biocompounds will vary greatly between different environments, and the specific nature of such molecules in any setting will inevitably alter over time.
What is needed in order to deal with this? One might consider a system where each potential threat was countered and nullified by a specific recognition molecule, but this proposal soon looks quite impractical if a very large number of potential molecular agents are possible. Also, as just noted, any such agents are not fixed immutably – and even a small chemical alteration might stymie effective recognition by a specifically-directed receptor. Immune systems facing challenges from infectious biological replicators have used a variety of strategies, culminating with adaptive immunity where complex mechanisms are used to generate receptors which are indeed ‘tailored’ to a new and novel threat. This level of sophistication has never evolved for dealing with non-replicating chemical poisons, an issue to be revisited in a subsequent post.
How then is defense against noxious chemicals obtained? While there is no comparable specificity to that seen with antibodies generated by adaptive immune systems, multiple lines of defense have evolved to counter specific poisonous threats. Dangerous levels of certain metal ions, for example, can be countered through the actions of proteins called metallothioneins, which bind and sequester such metals and thereby ameliorate their toxic effects. Strongly oxidizing chemical groups (whether generated through normal metabolic activities or acquired from the environment) are routinely mopped up by a variety of endogenous antioxidants, among which the versatile metallothioneins are included. But of particular interest in this context is the huge diversity of foreign organic small molecules which might potentially impact upon any organism’s normal biological operation – how can these be effectively neutralized?
Before looking at this question, it would be useful to consider a little semantics revolving around what will become a key word here: ‘xenobiotic’. This word, literally ‘stranger to life’ is often used in two distinct, though overlapping, senses. Firstly, it can refer to any molecule which is foreign to the physiological functioning of an organism in question. In other words, in this sense a ‘xenobiotic’ denotes any molecular entity which is not synthesized by the organism itself, or which is not normally used by it as a food, nutrient cofactor, or for any other function. As such, it covers the whole gamut of natural products deriving from the collective biosphere which are foreign to the normal make-up or functioning of a specific organism. Clearly, though, this definition would also encompass all artificial molecular entities, all molecules whose origins derive entirely from human ingenuity. And here is the second sense of ‘xenobiotic’ arises, since in many cases this word is used to refer (more or less) exclusively to artificial compounds, especially where they have become environmental contaminants. Although this framing of the word is more restrictive, it would seem actually closer to its literal meaning as foreign to life in general, thus indicating something truly new under the sun. Nevertheless, for the present purposes it will be used in the first sense, which embraces natural ‘foreignness’, as well as artificial sources of molecular ‘non-self’. After all, no-one suggests that defenses against foreign chemical agents evolved to deal with the advance possibility of non-natural compounds emerging in the environment!
Levels of xeno-defense
Since it is impossible for an organism to avoid taking in physical materials from its environment, the potential for exposure to noxious chemicals will always exist. But equally obviously, the risks from ingesting nutrients are not evenly distributed across the environment as a whole, and avoiding foci of possible danger is of clear value. This is simply in accord with the old dictum, ‘prevention is better than cure’, although of course blindly applied by favorable evolutionary selective pressures. For mobile animals, chemosensory perception has an important role in screening out noxious nutrient sources. Potentially dangerous decaying foods can warn via their odors, and many food sources (especially plant-derived) bearing toxic secondary metabolites signal the potential threat through strongly bitter and aversive tastes. Since most toxic plant alkaloids are not volatile, taste aversion is likely to be the most important means of primary screening of potentially noxious environmental compounds.
This then returns us to the above general question regarding how an organism can handle the multiplicity and diversity of potential molecular threats, by asking how the front-line taste-based screening can work. It is now known that the perception of bitterness is mediated by the ‘Type 2’ taste receptors (TAS2R), encoded by about 30 distinct genes in the human genome. Obviously this is massively insufficient to cover the scope of potentially noxious compounds, if each receptor was specific to a given target structure. While much more information is needed, it appears that while different TAS2R receptors respond to different bitter tastants, the receptors as a whole are not dedicated to unique structures. A key descriptive word in this context which will apply at other stages of xenorecognition is ‘promiscuity’, or relaxed discrimination between different molecular structures serving as recognition targets. Presumably, the promiscuity shown by the TAS2R receptors is sufficient for perception of a wide enough range of structures to be biologically useful as a front-line gating of potential poisons. (Each receptor is likely to have its own pattern of structural recognition, such that collectively the receptors cover a sufficiently adequate area of chemical space).
Clearly, though, other lines of defense against noxious molecules will be needed. While obviously biologically useful, gating against primary ingestion of poisons could not provide any guarantees. Toxic products might fail to register bitterness, be so potent as to be still dangerous when below the threshold of taste awareness, or be masked by other tastants present in the entire ingested material. Or poisons might be inadvertently taken in by non-oral routes, thereby circumventing anything that TAS2R signaling could achieve.
The conventional view of the processing of ingested drugs (meaning essentially the same as natural or artificial xenobiotics in this context) is divided into three metabolic phases, involving various Xenobiotic Metabolizing Enzymes (XMEs) and other factors. In Phase I metabolism, xenobiotics are acted on by enzymes (particularly those of the cytochrome P450 family) which incorporate or expose chemical functional groups, by redox or hydrolytic reactions. In Phase II, the initial processing facilitates the transfer of natural biological groups onto the xenobiotic to form various conjugates. Phase III reactions (those occurring post-conjugate formation) can involve further processing by Phase I enzymes, and often are taken to include the export of modified xenobiotics across cell membranes by various efflux systems.
Enzymes modifying ingested xenobiotics must clearly be capable of recognizing their molecular structures, although (as seen with the TAS2Rs above) not necessarily with high specificity. In relatively recent times, it has become apparent that before the onset of the Phase I metabolic processing, the primary recognition event involves key proteins generally termed ‘xenosensors’. Many of these had been previously discovered and defined as part of a nuclear receptor superfamily, but initially termed ‘orphans’ owing to their uncharacterized ligand-binding functions. Some such proteins, however, were later found to bind xenobiotic compounds, an interaction which in turn activates these nuclear receptors as transcription factors regulating the expression of key downstream Phase I-III proteins. (This new knowledge accordingly released these nuclear receptors from their ‘orphan status’). Among these xenosensors, the pregnane X receptor (PXR) and constitutive androstane receptor (CAR) have received much attention, but various other xenosensing nuclear receptors exist.
Another important xenosensor is the aryl hydrocabon receptor (ArHR), which is initially located cytoplasmically in distinction to the above nuclear receptors, although upon binding one of its target ligands, the ArHR is then translocated to the nucleus for regulation of its specific transcriptional targets. The figure A below depicts both primary xenosensing and the above-noted three Phases of xeno-processing:
Figure A. Depiction of cellular recognition for a hydrophobic xenobiotic (able to directly traverse cell membranes). Primary xenosensing, and the three Phases of metabolic processing are depicted, culminating in the export of modified compound. This simplified depiction does not attempt to show subcellular locations of the various metabolic components. The xenotransporters can act on conjugates between modified xenobiotics and ubiquitous factors such as the peptide glutathione, but in some cases xenobiotics may be directly exported. (These alternatives represented by the xenobiotic in red).
Figure B. Depiction of xenobiotic recognition for protective purposes as a process where the front-line is held by odorant and (particularly) taste receptors.
The manner in which xenobiotics are metabolized is crucial to the outcome of the exposure of an animal to the alien molecule(s). All of the players in xenobiotic responses and handling can vary genetically, and this can be a major influence on outcomes for both the short and long-term.
Genetics and Poisons
In passing, we can note that genetic variation in response to chemical challenges is not limited to organic compounds. In the first post of this ‘subtle poison’ series, the deleterious effects of both heavy metals and mineral fibers was noted. In both of these cases, genetic influences on host responses have been recorded, although more data is needed to fully characterize the relevant genes involved in each area.
The role of individual variation in xenobiotic-metabolizing enzymes, and in turn variation in the way such molecules are processed between different individuals, has become of great interest in recent times. For the pharmaceutical industry and medicine, clearly an ability to accurately define how a drug will behave in a specific individual would be immensely valuable, and much useful information has been gained in specific cases. In particular, studying differences in cytochrome P450 family allelic enzyme activity levels has been a profitable undertaking, with clinical applicability.
But for the present purposes, let’s look at particular aspect of the innate genetically encoded anti-xenobiotic responses, where the response itself is responsible (wholly or in part) for the resulting toxic effects.
An Autoimmune Analogy
Earlier in this post, the response against xenobiotics was contrasted with immune systems evolutionarily selected for countering infectious replicators. A fundamental difference between the vertebrate adaptive immune system and responses to xenobiotic threats is the restriction of the latter to sets of germline genes. In other words, while adaptive immune systems can generate and select novel receptors for countering previously unanticipated pathogens, the xenobiotic ‘immune system’ is expressed from innate sets of genomic coding sequences. In this respect, responses against ingested xenobiotics have more in common with the innate immune systems (of either vertebrates or invertebrates), where gene products recognizing specific microbial ‘danger signals’ are encoded in germline genomes.
The adaptive immune system’s greatest strength, the generation of novel receptors to meet novel threats, is also a potential source of harm through the unwanted generation of self-reactive immune specificities. Even though evolution has developed extremely sophisticated ways of avoiding this, adaptive immune system autoimmunity presents an ongoing clinical burden. It might be thought that any innate defense system would bypass this problem, since any innately encoded proteins or nucleic acids recognizing self should be strongly selected against through evolution. Yet it is now known that certain aspects of innate immunity can indeed help trigger autoimmunity under specific circumstances.
Responses mediated by xenobiotic sensors and processors can also directly mediate deleterious results, in contradistinction to the ‘proper’ physiological roles. Although there is no direct parallel with innate immunity to be made, certainly one can view such inadvertently self-destructive responses as ‘autoimmune’ in a broad analogous sense, if one likewise considers xenobiotic processing as a special kind of innate (and usually protective) immunity in its own right.
Self-activation of xenobiotic deleterious effects
There is more than one pathway by which innate mechanisms can produce deleterious reactions to xenobiotic challenge. There is much precedent for the toxicity of a primary xenobiotic not being manifested until modifications in vivo by Phase I metabolic enzymes are produced. As a case in point, note that a previous post in this series looked at the generation of a neurological condition recapitulating Parkinson’s disease by the compound MPTP. Here the initial molecule was not the direct villain, but rather an MPTP derivative (MPP+) produced by the action of monoamine oxidase (MAO) enzymes. (A striking confirmation of this in animal studies is the blocking by MAO inhibitors of neuronal destruction otherwise mediated through MPTP administration). Also, a previously noted environmental xenobiotic chemical found in soot and coal tar, benzo[a]pyrene is modified by Cytochrome P450 enzymes to an active epoxide derivative, which directly forms DNA adducts ultimately contributing to its carcinogenicity. In these circumstances the Phase I enzymes therefore actually aid and abet the carcinogenic process.
Detoxification may require a sequence of enzymatic modifications upon an initial xenobiotic exposure. During this process, an elevated toxicity of intermediate derivatives may be ‘acceptable’ if their presence is transient and the overall chain of modifications leads to complete elimination of the initial toxic problem. Genetic variations in the activities of key enzymes which retard the removal of highly toxic intermediates could clearly result in significant problems. A classic exemplar of these processing factors is the metabolism of ethyl alcohol (ethanol). This widely popular compound is initially converted into acetaldehyde by alcohol dehydrogenase (and also the Cytochrome P450 member CYP2E1), then into acetate by aldehyde dehydrogenase, and ultimately into carbon dioxide and water. These aspects of ethanol metabolism are common to all humans, which means that anyone imbibing alcoholic beverages is exposed not only to ethanol itself, but also the same metabolic products. The most potentially dangerous of these is acetaldehyde, a known carcinogen. But since quite benign acetate results from the processing of acetaldehyde itself, the derivation of the latter from ethanol is transitory. But it may be a crucial issue just how transitory that effect should happen to be.
It would seem obvious enough that a variant of aldehyde dehydrogenase (ALDH2) with reduced activity would allow the build-up of acetaldehyde after ethanol intake, and this is indeed the case for a significant fraction of humanity (mainly in East Asia) bearing an allelic variant of this enzyme (ALDH2*2) with very low activity. Blocking removal of acetaldehyde renders the effects of alcohol unpleasant, a feature which can be produced in anyone by means of drugs inhibiting the ALDH2 enzyme. (This has been the basis of one type of treatment for alcoholism). But increased levels of acetaldehyde can also result if the catalytic rate of alcohol dehydrogenase (ADH) itself is higher than the usual baseline, and this is seen with the ADH allele 1C*2. In such circumstances, the elevated rate of acetaldehyde production (relative to its enzymatic removal) increases its transient concentration in comparison to that seen with normal ADH.
By whatever means increased levels of acetaldehyde may be produced, the same trend towards increased carcinogenicity results, and evidence for the role of acetaldehyde in ethanol-induced cancers is well-characterized. Under natural circumstances, alcohol may be ingested in relatively small amounts sporadically (think of fermented fruits), but high-level or prolonged exposure in humans is almost always through voluntary actions. So alcohol could be viewed as having ‘autotoxic’ effects involving both conscious-level decision-making, and also at the molecular level from an individual’s own metabolic processing enzymes. Acetaldehyde toxicity resulting from ethanol intake can also have both immediate effects (sickness, flushing) and more subtle long-term negative consequences (induction of tumors). And (to invoke the analogy with autoimmunity), some individuals are highly sensitive to the effects of acetaldehyde produced from ethanol directly as a result of their genetic backgrounds (as with the ALDH2*2 or ADH 1C*2 alleles).
Pursuing this theme a little further, it’s interesting to consider (as mentioned above in passing) that acetaldehyde can also be produced from ethanol through the Phase I metabolic enzyme CYP2E1. Normally, though, the contribution of CYP2E1 is small except in the case of heavy habitual drinkers, where the enzyme becomes induced. But alcohol is certainly not the only target for this enzyme, given the promiscuous range of substrate recognition by Phase I metabolic catalysts. It eventuates that CYP2E1 converts the common analgesic drug acetaminophen (paracetamol) into toxic derivatives, and when high CYP2E1 has been induced, serious liver toxicity can result from taking normally innocuous acetaminophen doses. Here, then, is another link between a higher-level behavior (albeit pathologized by alcohol addiction) and a ‘blind’ molecular process, both of which elicit ‘autotoxic’ effects.
In mice, the effects of CYP2E1 can be dramatically documented with gene knockouts. Removal of CYP2E1 activity by genetic ablation greatly reduces acetaminophen toxicity. Toxicity for another one of its substrates, benzene, is similarly removed, whereas normal mice given comparable benzene doses are severely affected.
Xenobiotics and Induced Receptor Activity
Now to consider a different pathway for self-inflicted deleterious effects from xenobiotics. Here the focus will be on the highly toxic compound 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), very often simply referred to as dioxin (although ‘dioxin’ per se is not chemically specific, and is also used to refer to related compounds.
Structure of TCDD
The combination of very high toxicity, environmental persistence, and generation as an unwanted industrial by-product render TCDD as a special problem. It came into particular prominence following the major contamination and human exposures resulting from the chemical plant accident at Seveso, Italy in 1976. Another boost to the notorious reputation of this compound came in 2004 from its use in the deliberate poisoning of the former president of the Ukraine, Victor Yuschenko, evidently an assassination attempt. (It is a little ironic that he is the second individual living within the former territory of the Soviet Union to be featured in this series of posts as a notable victim of a malicious toxic assault. The other person of note was the Russian Alexander Litvinenko, who succumbed to radioactive polonium, as noted previously).
TCDD exerts its effects through binding to the above-mentioned aryl hydrocarbon receptor (ArHR), resulting in its prolonged activation and pathological expression of ArHR target genes (when the receptor is translocated to the nucleus and acts as a transcription factor). As a xenosensor (depicted in the Figure A above), the ArHR activates expression of various downstream xenobiotic-metabolizing enzymes, but TCDD is a poor target of them, being very slowly metabolized. Combined with its high fat solubility, this compound has very long persistence in humans, and its in vivo presence is thus associated with long-term over-activation of the ArHR.
Animal studies provide strong evidence that binding and activation of the ArHR by TCDD is its primary, if not exclusive, mechanism of toxicity. Mice with the ArHR gene ‘knocked out’ by gene targeting technology show a stunning resistance to TCDD-mediated effects. (Although the ArHR certainly has normal physiological roles, and animals lacking this receptor show certain defects, they can grow to maturity and thereby allow such toxicological studies to be conducted).
To refocus on the ‘autoimmune’ theme of this section, consider that in toxicology, it is commonly stated that ‘the ArHR receptor mediates dioxin toxicity’ (or in words close to that effect), reflecting the inescapable conclusions of the above knockout data and many other studies. Rather than a poison directly damaging the functioning of an organism, in this case the poison only creates havoc by effectively enlisting a host factor to initiate self-harm. Consider also that if the ArHR is a component of a system with an important role in detoxifying xenobiotics, then for TCDD (and other known chlorinated polycyclic compounds) the process is subverted towards a biologically dysfunctional end. As such, the TCDD / ArHR precedent would appear to be a classic exemplar of the analogy of ‘misfired’ xenobiotic responses with autoimmune reactions.
Xenobiotics and Real Autoimmunity
Although TCDD and the ArHR are thus used to exemplify a self-damaging process analogized with autoimmunity, ironically they also provide a very cogent link with autoimmunity which is real in every sense. Evidence suggests that xenobiotics can induce autoimmunity by at least two processes, somewhat reminiscent of the above two pathways for xenobiotic-induced self-damage itself. There is now considerable experimental data supporting the contention that self-proteins which have become modified by reaction with xenobiotic compounds (‘modified self’) can trigger immune reactions which cross-react with normal self-structures, and thereby trigger an autoimmune response. This kind of effect has often been termed ‘molecular mimicry’ elicited by the xenobiotic-derived host neoantigens. Alternatively, modification of host proteins by foreign chemicals may generate self-recognition of otherwise cryptic self-epitopes. Exposure to certain heavy metals (mercury in particular) can also trigger unequivocal autoimmunity in animal models, probably by similar mechanisms.
Theoretically, a second broad means of physiological modulation by xenobiotics which might lead to autoimmunity could be differential effects on specific immune cellular regulatory subsets. Real evidence towards this comes from the TCDD / ArHR system once more. It turns out that a special effector helper T cell subset (TH17) bears the ArHR receptor, and prolonged signaling induced by exposure to TCDD activates these cells and exacerbates the development of autoimmune disease in mouse models. (Knockout mice lacking the ArHR accordingly lack this TCDD-induced susceptibility to autoimmunity).
So it seems indisputable that there are good grounds for proposing real intersections between xenobiotic processing, its perturbation, and autoimmune phenomena. And there we leave it for the time being. A point to consider in the next post is why some xenobiotics trigger actions which result in self-damage rather than clear detoxification.
To finish, here are two biopoly(verse) offerings. The first is made with respect to genetic influences on xenobiotic recognition, while the second refers to self-damaging responses to xenobiotic challenge:
People say, ‘So choose your parents well’
For your genotype surely will tell
How well you survive
And prosper and thrive
In a toxicological hell.
The war against toxic attrition
Is a physiological mission
But within this good fight
There are factors that might
Link self-harm as a point in addition.
References & Details
(In order of citation, giving some key references where appropriate, but not an exhaustive coverage of the literature).
‘…..proteins called metallothioneins….’ These proteins (of which there are several classes) also have roles in the transport and delivery to specific subcellular sites of metal ions required for normal metabolic function. Metallothionein-mediated protection against metal ion toxicity is best characterized in the case of cadmium, but is also implicated in protection against mercury and possibly lead toxicities. For more detail see Klaassen et al. 2009; Sutherland & Stillman 2011; Gonick 2011.
‘……a variety of endogenous antioxidants…..’ These include Vitamins C and E, glutathione, and numerous others. For a review, see Rizzo et al. 2010.
‘…..the perception of bitterness is mediated by the ‘Type 2’ taste receptors….’ See Behrens & Meyerhof 2009.
‘…..different TAS2R receptors respond to different bitter tastants….’ In this respect, see an article about a database of compounds with bitter taste (Wiener et al. 2012), one of whose aims is to promote the understanding of the recognition of target molecules.
‘ Each [bitter taste] receptor is likely to have its own pattern of structural recognition….’ See Meyerhof et al. 2010.
‘….the processing of ingested drugs …..is divided into three metabolic phases…..’ For aspects of these phases, see Nakata et al. 2006.
‘….. the pregnane X receptor (PXR) and constitutive androstane receptor (CAR)….’ See a review of Tolson & Wang 2010.
‘….other xenosensing nuclear receptors exist.’ These include the peroxisome proliferator-activated receptor (PPAR), the farnesoid X receptor, and hepatocyte nuclear factors (1alpha, 3 and 4alpha). See Dixit et al. 2005; Xu et al. 2005.
‘…..the aryl hydrocabon receptor.…’ For a general background, see Abel & Haarmann-Stemmann 2010.
‘…..the deleterious effects of both heavy metals and mineral fibers was noted. In both of these cases, genetic influences on host responses have been recorded….’ For the heavy metals mercury and lead, a number of genes have been implicated (see Gundacker et al. 2010). In the case of mineral fiber-related diseases (especially mesothelioma caused by asbestos), it was noted in a previous post that cofactors were certainly involved. A genetic predisposition towards mesothelioma resulting from another mineral fiber (erionite) has been identified through family studies in Turkey (Dogan et al. 2006; Below et al. 2011).
‘ It might be thought that any innate defense system would bypass this problem [autoimmunity]….’ As an example of this point of view made before contrary evidence emerged, see Medzhitov & Janeway 2000.
‘….certain aspects of innate immunity can indeed help trigger autoimmunity under circumstances.’ Without going into too much detail, this can involve circumstances where specific types of innate recognition are controlled by cellular compartmentalization, and its perturbation in pathological states. A little more is provided in the adjunct ftp site (Extras; Chapter 3, Section A3) for Searching for Molecular Solutions. See also Rai & Wakeland 2011.
‘…….benzo[a]pyrene is modified by Cytochrome P450 enzymes to an active epoxide derivative…….’ See Ling et al. 2004.
‘….and finally into carbon dioxide and water….’ This occurs via the formation of acetyl-CoA and the citric acid cycle, described in any standard biochemistry text.
‘…..acetaldehyde, a known carcinogen….’ / ‘….ALDH2*2 ….. the ADH allele 1C*2.‘ / ‘….the role of acetaldehyde in ethanol-induced cancers is well-characterized.’ For more background information on these topics, see Visapää et al. 2004; Seitz & Stickel 2009.
‘……..drugs inhibiting the ALDH2 enzyme [have] been the basis of one type of treatment for alcoholism….’ The classic drug in this regard is disulfiram, although the merits of its use are still controversial. See (as an example from a large literature) Jorgensen et al. 2011.
‘…..some individuals are highly sensitive to the effects of acetaldehyde produced from ethanol directly as a result of their genetic background (the ALDH2*2 or ADH 1C*2 alleles )…..’ A very interesting recent development is the observation that the ALDH2*2 mutation results in incorrect protein folding, a defect which can be corrected by a low-molecular weight ‘chemical chaperone’. (See Perez-Miller et al, 2010) Thus, in the near future perhaps enforced non-drinkers may become capable of imbibing alcohol by co-use of drugs assisting their endogenous defective aldehyde dehydrogenase enzymes, although it’s possible that not everyone would agree that this is a good thing.
‘……chemical plant accident at Seveso, Italy in 1976…..’ For more on this, see an online article from Time magazine. The accident was clearly associated with many cases of a skin disorder caused by dioxin (chloracne), but the effect of dioxin exposure on cancer rates in the exposed Seveso population have been controversial. In this regard, see Boffetta et al. 2009. (TCDD is clearly carcinogenic in animal models).
‘…..the deliberate poisoning of the former president of the Ukraine, Victor Yuschenko….’ Despite bearing massive amounts of TCDD, Yuschenko survived, albeit with severe chloracne, with his symptoms slowly improving over several years. His clinical profile has been studied and reported (see Sorg et al. 2009). Unless the intention is to simply cause great pain, discomfort and disfigurement, TCDD would seem a foolish choice for malicious poisoners. Unlike rodents and other mammals, humans are not particularly susceptible to lethal effects from TCDD. Also, its overt clinical manifestation (chloracne), its in vivo persistence, and its ready detection render intoxication with TCDD easily proven.
‘ Mice with the ArHR gene ‘knocked out’ ….. show a stunning resistance to TCDD-mediated effects.’ For a review of such studies, and other xensosensor knock-outs, see Gonzalez et al. 1995.
‘…..the ArHR certainly has normal physiological roles……’ See Abel & Haarmann-Stemmann 2010 for background information on ArHR biology.
‘……self-proteins which have become modified by reaction with xenobiotic compounds …… thereby trigger an autoimmune response.’ Although protein modifications by xenobiotics have been known for over half a century, much research in the past few decades focused on DNA chemical adduct formation, given the obvious link in such cases with mutation and aberrant DNA processing or replication. More recently, it has become clear that protein damage too can have grave pathological consequences, of which autoimmunity is a significant part. The study of xenobiotic-mediated protein adducts has greatly benefited from recent advances in proteomic technology. See Liebler 2008 for a detailed exposition of these matters.
‘…..Exposure to certain heavy metals (mercury in particular) can also trigger unequivocal autoimmunity….’ See a review by Schiraldi & Monestier 2009.
‘…..a special effector helper T cell subset (TH17) bears the ArHR receptor, and prolonged signaling induced by exposure to TCDD …… exacerbates the development of autoimmune disease…..’ For more on this (and other aspects of TCDD effects on immunity) see Veldhoen et al. 2008; Esser et al. 2009.
Next biopolyverse offering to be posted in May, given current commitments.
The theme of the previous post concerned how human diseases could be triggered by environmental compounds with slow and subtle effects, with an emphasis on those which occur naturally. (The interest in natural exemplars of such effects arises from earlier posts on ‘Natural Molecular Space’). A principal theme in this follow-up post will be comparing cancer and cellular degeneration induced by environmental agents.
Subtle Carcinogens and Other Problems
With the exceptions of Polonium-210 and asbestos, the ‘subtle poisons’ considered previously were neurotoxic organic molecules. But organic cancer-causing compounds have been described for a long time. The first description of an association between a specific cancer and an industrial (work-related) activity dates back to the 18th century, when a rare form of scrotal cancer was linked to chimney sweeping. From the time of publication of this finding, it took almost 160 years for science to advance enough such that the active component in soot and coal tars was identified as benzo[a]pyrene, a polycyclic aromatic hydrocarbon. Certain other such polycyclic aromatics are also carcinogenic, and they are thus known collectively as PAHs.
Of course, we now know that a whole zoo of both natural and artificial compounds can induce cancer, with varying degrees of potency. It isn’t the intended scope of this post to review a great number of specific cases here, but among the natural set of known carcinogens, an important group are derived as secondary metabolites of various fungal organisms (metabolic products which are not essential components of fundamental life-support processes). While some secondary metabolites (such as antibiotics) have been extremely beneficial to humans, a ‘dark side’ of such secondary metabolism also exists. Not all toxic fungal products (or mycotoxins) are proven carcinogens, but some most certainly are. Probably the most significant in economic and human disease impact are a group of closely related compounds called aflatoxins, produced by various species of the fungal Aspergillus genus (most notably A. flavus). Aflatoxin B1 is the most potent known natural liver carcinogen, and a major problem as a side-effect of fungal contamination of foodstuffs, such as peanuts.
Sometimes it is the case that carcinogens are not directly found in certain natural food materials, but are actually formed during cooking processes. There is a certain irony here, because on the whole, cooking of many foods is beneficial through the killing of potentially dangerous parasites, especially those harbored in raw meat. And apart from the generally detrimental effects of parasites on health, a number of such organisms are themselves directly linked to the generation of specific cancers. Yet during ordinary cooking of meats, carcinogenic heterocyclic amines can form, and if charring is involved (as with barbecuing), polycyclic aromatic hydrocarbons can be created. Among the latter is found benzo[a]pyrene, the same compound of chimney sweep fame as noted above. Strictly speaking, carcinogens formed by cooking are not ‘natural’, since they require human intervention for their formation. Indeed, cooking itself has been considered a useful marker for distinguishing humans from all other organisms, including our primate relatives, and may have even shaped evolutionary pathways leading to modern humans. Still, while carcinogenic compounds resulting from cooking clearly arise from human agency, their formation has always been completely inadvertent, and occurred long before the faintest glimmerings of modern chemistry.
Subtlety of effect, at least as measured by the time between exposures and onset of disease, is practically a by-word for carcinogens, as well as the ‘subtle’ neurotoxic agents considered in the previous post. This is not to say that these two broad areas of pathology cover everything where subtlety rears its head, but they may safely be grouped as the major concerns. Beyond this, one needs to consider other physiological systems which may be damaged or negatively affected slowly and subtly by non-biological environmental agents, but not with tumorigenic outcomes. One case in point is the immune system, and there are precedents for natural compounds with immunosuppressant qualities. In this respective, it should be noted that toxic compounds can have multiple effects, and aflatoxins (for example) have immunosuppressive activity as well as their other noxious manifestations. Reproductive systems can be adversely affected by natural phytoestrogens, as considered in more detail in a previous post .
These other issues aside, cancer and toxic neurological disease can be seen as book-ends in terms of the gross effects leading to divergent pathological results. Let’s consider this statement a little further.
Growth or Degeneration, and a Problem Either Way
A toxic challenge will by definition perturb normal cellular functions. Following such an event, broadly speaking three things can happen. Firstly, an affected cell may, through its endogenous repair system, correct the damage and resume its normal functions. Failing this (in the second alternative) it can die, through a number of alternative mechanisms noted in the legend to the figure below. The best-defined form of directed or ‘programmed’ cell death is the process termed apoptosis. But if death itself should fail and replication continue, chromosomal changes induced in the cell may eventually lead to ‘transformation’, where the normal controls on growth are circumvented and a tumor phenotype acquired, the third possible outcome. Successive genetic changes can accumulate, and transformed cells with invasive properties become amplified through their enhanced growth and survival properties. It is no accident that important genes regulating apoptosis are frequently mutated in cancer cells. If checkpoints on cell growth are removed through blockade of cell death, barriers to transformation may be greatly reduced. Indeed, while most carcinogens are also potent mutagens (inducing genetic mutations in genomic DNA), some are not. The latter have been a long-standing puzzle, but it has been shown that non-mutagenic chemical carcinogens are direct blockers of apoptosis, thereby allowing cells with mutations (normally removed by apoptosis) to persist and proceed down transformation pathways.
As noted in the previous post, recovery from a toxic insult might not necessarily be complete, in the sense that the post-toxic state may be sub-optimal relative to the norm, predisposing the cellular victims to future risk. But leaving such complications and the general area of damage repair aside, the major enduring pathological consequences of a low-level toxic assault revolve around cancer vs. degeneration. These outcomes might seem like diametrically opposed processes, since in one case cells grow wildly without normal constraints, and in the other, they die. While the final end-points are clearly quite divergent, it is interesting that the factors which push cells along these pathways have many regions of overlap. Genetic analyses have shown that many mutations which predispose towards Parkinson’s disease are also associated with certain cancers.
But toxic chemicals can also have dramatically different effects depending on the cellular context in which they act. A specific genotoxic (DNA-damaging) compound found in cycad plants (methylazomethanol) can induce neurological damage and degeneration in mice without tumor formation, whereas a high frequency of tumors are induced in the colon. Major determinants of the outcome of such toxic challenge are the levels of appropriate DNA repair enzymes (the effectiveness of the DNA damage response), and differential effects on cellular signaling pathways. Up- or down-regulation of specific pathways operating in diverse cell lineages can evidently result in outcomes as distinct as death or degeneration. A clear distinction between neurons and most other differentiated cells is their cell division status, where non-dividing and long-lived neurons can be contrasted with lineages with active turnover through cell division. Neurons thus permanently exit from the cell cycle into a ‘post-mitotic’ state for the lifetime of the organism.
Indeed, trying to force a mature neuron towards re-entering the cell cycle (by artificially expressing viral gene products which ‘kick-start’ cycling in other quiescent cells) has been observed to promote cell death. Given this piece of information, a differential response to at least some toxic agents can conceptualized in fairly simple terms: Forcing a quiescent cell which is nonetheless ‘primed’ for mitotic cycling (active division) may lead to carcinogenic transformation; doing the same thing to a mature neuron will kill it. This dichotomy is portrayed in the figure below:
Outcomes of mutational damage through low-level genotoxic exposure for neurons vs. non-neuronal cell lineages. In both cases, repair mechanisms exist, which may be insufficient to deal with the problem. Dividing cells may then be diverted into a programmed cell death pathway (usually apoptosis) and thus removed. In a population of renewable replicating cells, this is unlikely to be a direct problem, and of course eliminates a potentially dangerous altered cell. Yet if the shunt towards apoptosis fails for any reason, the altered cell may continue to proliferate and acquire further mutations, with the ultimate consequence of malignant transformation into a fully cancerous phenotype. For neural cells, beyond a certain damage threshold, death is inevitable, even for stimuli that normally promote mitosis in other cell types. Note here that cell death in general can occur by at least three mechanisms, shown specifically for neurons in this schematic. The process of autophagy (a kind of self-recycling of cellular components) is associated with repair processes, but can also constitute a specific cell death mechanism in some circumstances. Apoptosis is a programmed form of death operating through specific cellular signaling interactions, and autophagy can interface with some of these apoptotic pathways (as shown by arrows). Necrosis was originally categorized as non-specific and passive cell death brought on by severe physical or chemical insults. While cell death in such a disordered manner is presumably still a possibility, recent evidence has shown that at least one form of non-apoptotic ‘necrotic’ cell death is also a regulated process, which has been duly termed ‘necroptosis’. In any event, unlike loss of cells with a high mitotic turnover, death of non-dividing neurons will ultimately have significant functional consequences.
And yet the above figure might, upon further reflection, appear overly simplistic. Two pieces of extra widely-known information are relevant here: neurological tumors, and neurological plasticity. If neurons die upon transformation, what is the source of brain tumors? And what about the considerable publicity which has been given to the previously unsuspected potential for recovery from substantial brain damage, indicative of ‘neuroplasticity’? In both cases, a simple answer is that neither the ‘dark side’ of neural tumors nor much brighter prospects for neural regeneration derive from irrevocably post-mitotic cells. In both cases, neural stem cells, identified only relatively recently, may be the central players. With respect to tumors, the “neural stem cell hypothesis” proposes their origin as the source of primary brain tumors, as opposed to metastatic tumors which originate elsewhere but migrate to the brain and grow there.
The above figure addresses genotoxic substances, but this does not include other neurotoxic agents such as the compound MPTP, discussed in the previous post owing to its specific role in inducing cell death in the substantia nigral brain region, and thereby leading to induced Parkinson’s disease. As noted previously, MPTP has a distinct toxic mechanism via the enzymatic formation of a specific metabolic product, which is taken up by dopamine-producing neurons. This metabolic derivative then inhibits mitochondrial respiration, leading to cell death. Studies have found, however, that MPTP also has mutagenic properties – or at least, once again, one of its metabolic products is the active compound in such assays. Yet even if MPTP indirectly caused unrepairable genomic lesions in target neurons, the above observations suggest that cell death would still be the case anyway, rather than prolonged growth and transformation.
To conclude then with a summary of sorts upon this theme:
It seems cancer and neural decay
Are opposed, in a particular way
For to die or to grow
Is the question, you know
And the source of young Hamlet’s dismay
References & Details
(In order of citation, giving some key references where appropriate, but not an exhaustive coverage of the literature).
‘ ….an association between a specific cancer ….. chimney sweeping‘ This resulted from observations by Dr. Percival Pott (1714-1788) for scrotal cancer in young chimney sweeps, first published in 1775. See Brown & Thornton 1957 for relevant historical information.
‘….the active component in soots and coal tars was identified as benzo[a]pyrene….’ See Ling et al. 2004 for more information, especially including the structure of benzo[a]pyrene-DNA adducts, by which its carcinogenicity is manifested.
‘…..a whole zoo of both natural and artificial compounds can induce cancer…..’ For a review on the diversity of carcinogens (including not limited to organic compounds), see Yang 2011.
‘……toxic fungal products (or mycotoxins)…..’ For a useful review, See Pitt 2000.
‘Aflatoxin B1 is the most potent known natural liver carcinogen….’ See a review by Hedayati et al. 2007.
‘…..a number of different parasites are themselves directly linked to the generation of specific cancers….’ If we consider ‘parasites’ in the broadest sense, then there are numerous precedents of viral and bacterially-generated cancers. But in food-related circumstances, ‘parasite’ will most often refer to various worms, some of which are indeed associated with cancer. For example, see Vennervald & Polman 2009 for a review of the status of helminth worms as carcinogenic agents.
‘…..if charring is involved……polycyclic aromatic hydrocarbons can be created….’ See Daniel et al. 2011.
‘……cooking ….. may have even shaped evolutionary pathways leading to modern humans…’ The ‘cooking hypothesis’ has lead to a very interesting book by Richard Wrangham, Catching Fire: How Cooking Made Us Human; Basic Books 2009.
‘……there are precedents for natural compounds with immunosuppressant qualities….’ Although in normal circumstances immunosuppression is obviously undesirable, for some medical applications certain natural immunosuupressants have proved a great boon. Such compounds have proven highly useful for suppressing unwanted immune rejections of transplanted organs, and thereby greatly facilitated the efficacy of transplant surgery in general. These include cyclosporin A and FK506, which form ternary complexes between cellular proteins (cyclophilin and FKBP respectively) and the protein phosphatase calcineurin. See Fox & Heitman 2002 for a review.
‘……while most carcinogens are also potent mutagens…….some are not….’ See Kokel et al. 2006, for presentation of evidence that specific non-genotoxic carcinogens act by suppressing apoptotic cell death.
‘…..many mutations which predispose towards Parkinson’s disease are also associated with certain cancers….’ See Devine et al. 2011.
‘……found in cycad plants…..’ See the previous post for more detail regarding the neurotoxic effects of cycads.
‘A specific genotoxic (DNA-damaging) compound found in cycad plants (methylazomethanol) induces neurological damage ….’ This refers to an interesting publication of Kisby et al. 2011.
‘…..cell death…..’ For further information regarding autophagy, see Kaushik & Cuervo 2006. For a perspective on apoptosis in the light of the recently described necroptosis, see Christofferson & Yuan 2010.
‘……publicity which has been given to the previously unsuspected potential of recovery from substantial brain damage….’ Neuroplasticity has received much popular notice largely owing to the book The Brain That Changes Itself, by Norman Doidge. Viking, 2007. Note that one aspect of neuroplasticity, the ability of neurons to exhibit ‘regenerative sprouting’ from axons, is not the same as acquiring the ability to undergo full cell division. See Weiloch & Nikolich 2006.
‘…..the “neural stem cell hypothesis” proposes their origin…..’ For more information, see Germano et al. 2010.
Next Post: Late March.
The past series of posts have largely been preoccupied with the benefits to be had from ‘natural molecular space’, whether the molecules in question are large, small, or functionally linked together in complex (but useful) entire biosystems.
Obviously, some biomolecules are not merely useless, but may be actively harmful. There are a great many bioproducts which are of both high toxicity and obvious impact, at least to the unfortunate victims of serious or even life-threatening natural poisonings or envenomations. But toxic effects can be much more subtle, and therefore much less easily noticed. In fact, the insidious slowness of some toxic effects can render the actual molecular culprits very hard to pin down, and inevitably controversy is thus generated. These ‘subtle negative’ environmental influences are the principle theme for this discussion, which will include natural products, but will also heavily feature both artificial compounds and non-biological but ‘natural’ substances. (The quotation marks are used here since it is very often only through human activities that natural materials with potentially harmful effects are processed and brought into contact with sizable numbers of people).
What Does Subtlety Mean in a Toxic Context?
When we speak of a subtle toxic effect, what is actually meant? It might result from several factors, or any combination of them, including potency, exposure dose, frequency of exposure over time, and the in vivo persistence of the toxic substance. Any ingested toxic compound must by definition interfere with an important biochemical process, with ensuing negative consequences for the functioning of the organism. A poisonous substance might interact with many different biological molecules, but some of these will be of greater import than others in terms of how the resulting deleterious effects are produced. And the affinity of the poison for such biological targets is a determinant of potency.
Potency and dosage over time are inter-related. To qualify as ‘subtle’, intake of a highly potent compound (one whose toxic threshold is reached with very small amounts) would need to be in exceedingly low quantities, where no immediate effects are apparent. If that was the end to it, then obviously such a low-level exposure to the toxic agent has no further consequences. But a subtle deleterious effect might exist if the compound had produced some kind of persistent tissue or cellular damage, of a type that was very hard to detect without sophisticated intervention, and that was not at all appreciable by the individual concerned. Then, several possibilities could exist which in the end would result in a manifested disease state. Firstly, if the individual is re-exposed to the same source of the toxin on more than one occasion, the damage might be cumulative and accrete until it becomes of such significance that an overt illness is produced. If the body’s repair systems cannot comprehensively deal with the low-level induced damage, in some cases even long intervals between exposures might still result in noticeable pathology. But even if the repair is effective, regular intake of similar low doses of the toxic material over time might eventually overwhelm the host defenses, again leading to disease.
These scenarios assume repeated exposures, but even a single exposure could potentially have significant consequences. It might be supposed that a single bout of damage, if not fully repaired, might be another negative event in an individual’s ‘wear and tear’ list that increases with ageing. In other words, any such a low-grade but persistent toxic ‘insult’ might become more significant over time, in combination with other problems inevitably occurring through life. But a much more serious possibility also has been proposed, where short-lived exposure to certain chemical agents might actually set up an on-going pathological inflammatory process, even long after the original poison has been removed from the host system. This theme will be looked at in a little more detail in a later post in this series.
At this point, it’s very relevant to consider that there is an important issue relating to the physiological removal of toxic agents, or (in other words) how long it may be that noxious substances of any description can persist once taken into a host organism. Persistence has clear-cut implications for the ability of a substance to contribute to long-term and subtle deleterious effects. While water-soluble (hydrophilic) compounds are generally metabolized and excreted reasonably quickly, lipid-soluble (hydrophobic) compounds can be taken up by fat reserves and remain there for years, with only a slow diminution with time. A classic example in this regard is the insecticide DDT, whose tendency to persist in adipose (fat) tissue is well-described. Poisons which are themselves toxic elements obviously cannot be further ‘broken down’ chemically, and can persist through their interactions with normal biomolecules. For example, heavy metals such as lead and mercury can bind and inhibit numerous enzymes. Although the resulting complexes between metals and protein molecules may be physiologically degraded, release of the metal component may simply liberate it for another cycle of inhibition. In some cases, a noxious element may be physically or chemically similar to a normal biologically-used element, and replace it in certain biomolecules, with disastrous effects on metabolic activities. This is case for the toxic elements arsenic (capable of competing with phosphorus) and thallium (capable of competing with potassium).
Another major class of persistent and dangerous substances are certain mineral fibers, most notably asbestos. Poorly biodegraded long fibers (such as some mineral silicates, of which asbestos is a case in point) can persist indefinitely in specific anatomical sites. Although the mechanism is still incompletely understood, this can be associated with the generation of a chronic inflammatory process and ultimately carcinogenesis. The link between asbestos and mesothelioma is well recorded.
If we cast a wide enough net, another class of non-biological poisons must certainly be included: radionuclides, or radioactive isotopic versions of the elements. These can be either radioactive isotopic versions of normal elements of biological significance, or radioisotopes of non-biological elements. All such cases can be of either natural or artificial origins. Many examples of the former group can be cited, but potassium-40 (40K) is a natural radioisotope of interest, since it is contributes the largest portion of the radioactive background in living organisms. As such, it has been proposed as a major source of natural mutation, although experimental results have suggested that its contribution to mutation must indeed be (if anything) a subtle influence. Cases of relevant non-biological radioisotopes are likewise exceedingly numerous. Briefly, consider the example of polonium-210 (210Po), which can occur naturally, or can be generated by artificial nuclear reactions. This radioisotope is present in tobacco smoke, and it has been implicated as a major factor in the generation of smoking-induced cancer. Polonium-210 has also been in the news in recent years, through its use an exceedingly potent poison in the murder of the ex-Russian agent Alexander Litvinenko in London in 2006. There’s obviously nothing subtle about that, but as with any toxic agent, even polonium-210 can exert low-level effects if ingested in small enough doses. At that lower end of the exposure scale, the effects will vary among different individuals, but may contribute to cancers or other conditions, with an overall shortening of life expectancy.
Individual variation in responses to low-level toxic exposure reflect genetic variation in the metabolic processing of foreign compounds, or how the body reacts to the presence of noxious materials. There is much more to be said on this topic, which will be picked up at a later time within this series of posts. But for the time being, we can note this as one of a number of influences bearing on whether a low-level toxic exposure will have longer-term ‘subtle’ effects, depicted in the figure below:
A depiction of the range of various influences which can determine whether a substance could manifest a slow or insidious ‘subtle’ toxicity. Note that an implicit issue within ‘Generation of Ongoing Pathology’ is the ability of host systems to repair and contain toxic insults, as opposed to the generation of responses which are ultimately self-damaging.
The influence termed ‘cofactors’ in the above diagram simply refers to any other non-host factor which can interact with a proposed environmental toxic substance to exacerbate its action, or even be essential for the insidious toxic effect to be manifested in the first place. An interesting example is a putative requirement for the presence of simian virus 40 (SV40) for the generation of mesothelioma by asbestos.
For the rest of this post, I’ll move on to some specific examples of effects which have revealed subtlety in several senses of the word. The first case involves an artificial compound which is not strictly speaking an ‘environmental’ effect, since it required self-administration, if inadvertently. However, the experience with this compound has had many ramifications which do impinge on environmental influences, both man-made and natural.
(1) Parkinson’s Disease & Toxic Agents
In the early 1980s a remarkable series of events occurred which had implications across several fields of science and medicine. Although terrible and tragic in many ways, it provided a dramatic example of how a toxin can produce quite specific neurological effects, and had direct implications for the origins of Parkinson’s disease (PD). At that time in California, clinicians were confronted with a series of drug addicts in a state of ‘frozen’ mobility, which had many similarities to severe PD. Subsequent scientific detective work showed that this apparent similarity was more than just superficial. The sporadic condition of human PD is characterized by ongoing degeneration in a region of the brain called the substantia nigra, where destruction of neurons normally producing the crucial neurotransmitter dopamine leads to loss of muscular motor functions, eventually immobilizing the patient. These neurons are also pigmented, through the production of a type of melanin (‘neuromelanin’), an early observation which provided the name of this brain area (‘substantia nigra’ = Latin for ‘black substance’). A compound, L-dihydroxyphenylalanine (L-DOPA, which can access the brain and becomes metabolized to dopamine itself) can greatly alleviate symptoms, especially when first applied. The ‘frozen’ addicts likewise generally showed responsiveness to L-DOPA. By analyzing their common activities, the source of the problem was tracked down to their injection of a street drug preparation, a ‘synthetic heroin’, which in actuality was a botched attempt to make the drug meperidine (pethidine). The preparation that the clandestine chemists had produced contained sizable amounts of a different compound, N-methyl-4-phenyl-1,2,5,6-tetrahydopyridine (MPTP), eventually identified as the toxic culprit by means of animal testing. These studies also showed that MPTP ingestion resulted in specific damage to the substantia nigra, with associated loss of dopamine-producing neurons and the onset of parkinsonian symptoms.
Structures of some relevant molecules for the Parkinson’s / MPTP story. The amino acid phenylalanine is included as the precursor to dopamine, and to show its chemical similarity to L-DOPA. Meperidine is the drug towards which abortive synthetic attempts led to the formation of MPTP. MPP+ is the actively toxic metabolic product derived from MPTP itself.
The striking features of this story were widely reported in the scientific literature, and even found their way into popular fiction quite quickly. Those unmistakably victimized by MPTP had varying fates, ranging from death within a relatively short time, to survival for over a decade. But behind the initial cadre of severely affected patients, the prospect still remains of many more people developing PD from short-term exposure to MPTP (and initially subclinical damage) even decades ago. And this naturally raises one of the major implications of the whole MPTP saga: if a defined toxin can have such amazingly specific effects, could there not be other toxins in the environment with similar properties, which induce the neurodegeneration seen in ‘sporadic’ parkinsonian patients? In the course of these kinds of speculations, it was noted that the very description of this disease was a relative latecomer in 1817. Could the apparent lack of reporting of this disease in earlier times mean that ‘natural’ PD is actually a toxic condition, associated with the beginnings of the industrial revolution and newly introduced environmental pollutants?
Many studies have been conducted in order to evaluate this and related questions. In particular, exposure to certain insecticides has been a long-standing suspect as a potential agent of PD, but despite ‘probable cause’, this has not been firmly nailed down. These kinds of analyses must distinguish between genetic influences and environmental factors. (Many distinct genes are known to affect an individual’s susceptibility to PD, and this will be further considered in a subsequent post in this ‘subtle’ series). Studies with monozygotic (identical) twins illustrate this. In one detailed 1999 investigation, sets of monozygotic twins showed no significant differences in the concordance (common incidence in both twin pairs) of PD compared to non-identical twin pairs, but only (and this a crucial point) if the age of onset for either twin was after 51 years of age. Non-concordance of a disease in twin pairs in a controlled study is highly suggestive of environmental causes at least being contributing factors. Consider that if a disease does have a simple genetic origin, significant concordance would be expected in the (essentially) genetically identical pairs. Most cases of sporadic PD occur later in life, also consistent with (but far from proof of) a slow induction from environmental sources. But where PD does occur at younger ages, genetic influences (rare mutations, possibly in combination with environmental factors) might be postulated, and this is consistent with the higher concordance observed with identical twins with relatively young ages at the onset of PD. But the only general conclusion typically made at present is that the origin of sporadic PD is complex, with multiple genetic and environmental influences implicated directly or as suspects. And yet there is no question that, at least in certain genetic backgrounds, MPTP alone can induce a pathology with the key characteristics of PD. How does it do this?
A Stealth Poison At Work
Intensive studies on the mechanism of MPTP toxicity revealed that it was not the direct perpetrator of the neuronal damage. MPTP itself is acted upon by a specific enzyme within the brain, monoamine oxidase (MAO) B, which converts this compound into a positively charged species, the N-methyl-4-phenyl-pyridinium ion (MPP+, as shown in the above chemical structure figure). Consistent with this observation, inhibitors of MAO enzymes are protective against the effects of MPTP in animal models. MPP+ itself is capable of using the machinery for dopamine transport into neurons (using specific dopamine receptors), and this promotes its accumulation in very specific neuronal sites. It is important to note that this particular uptake mechanism also explains the high selectivity of MPTP (the precursor to MPP+) in its toxic action. Once taken up by dopamine neurons, MPP+ itself acts as a primary toxic agent towards mitochondria, through its inhibition of Complex I of the mitochondrial respiratory electron transport chain.
With the MPTP story, a series of processes are thus required for the ultimate toxic effect to be manifested: conversion to MPP+, uptake by dopamine neurons, and inhibition of mitochondrial activities. (These are primary factors; other issues such as specific genetic backgrounds certainly contribute to individual susceptibility, as will be discussed further in a subsequent post). So, it has been noted that this conjunction of requirements would (hopefully) render the occurrence of compounds with analogous properties to MPTP quite rare. With this in mind, are there natural precedents for this kind of noxious chemical agent? This raises the second case set to be considered (as noted above): natural toxic substances with ‘subtle’ actions. In many such circumstances, the subtlety is bound up with the difficulty of pinning down the true identity of the pathogenic culprit.
(2) Cycads, Soursops, and other ‘Environmental’ Neurological Diseases
In certain Western Pacific islands, epidemiologists have noted for decades an unusual incidence of a degenerative neurological condition called Amyotrophic Lateral Sclerosis / Parkinsonism-Dementia complex (ALS-PDC). In the language of the Chamorros of Guam, a people living on one of the afflicted island groups, the disease is known as ‘lytico-bodig’. A strong role for genetic influences in the origin of ALS-PDC seemed unlikely, given that it was recorded in diverse ethnic groups in varied Western Pacific locations. For a considerable time, though, a dietary item has been implicated: the consumption of a flour made from the seeds of cycad plants available in the affected locales. This remains unproven and controversial, particularly since a specific compound has not been conclusively identified. Yet the general ‘cycad hypothesis’ has support from a number of linked observations. Cycad flour fed to experimental animals over time induces a neurological condition with features of progressive parkinsonism, with associated damage to the substantia nigra. Also, the incidence of ALS-PDC has been in decline in recent years, and this correlates with changes in diet where the amounts of cycad-derived material have markedly declined. A specific amino acid, β-methylamino-L-alanine (BMAA; not found in normal proteins) has been repeatedly linked with cycad-induced disease, but proof of its role has consistently fallen short of the mark. Another contender is methylazoxymethanol (MAM, a metabolite derivative of the cycad compound cycasin), which has been shown to produce neurological genotoxicity.
Whatever the outcome of these studies, there is no question that raw cycad seeds (from which flour is derived) are quite poisonous, and this has long been known to Western Pacific peoples. But by using extensive washing and soaking procedures, they have ingeniously found a way to exploit this otherwise-useless material as a valuable foodstuff. The great irony implicit in the ‘cycad hypothesis’ is that although they succeeded in eliminating the acute toxicity of the cycad seeds, they could not remove traces of toxic substances which may have been the agents of subtle and insidious neurological damage.
Another potential natural molecular assailant of neurons is also found in an island setting, but in the West Indies. A high incidence of an ‘atypical’ parkinsonism has been identified on the island of Guadeloupe. (One example of the atypical nature of this condition is its failure to respond to L-DOPA.) This has been linked by epidemiological studies with the consumption of the tropical fruit called soursop, and a specific compound from this fruit (annonacin) has implicated as the probable underlying source of the pathology. Annonacin is an inhibitor of mitochondrial Complex I, and can also induce loss of dopamine neurons in the substantia nigra of experimental animals – findings which cannot help but stimulate recollection of the MPTP story, even if there are many points of divergence.
Finally, it’s interesting to note that both ALS-PDC of the Pacific and the Guadeloupe disease also have pathological features of ‘tauopathies’, or diseases associated with abnormal intercellular distribution of a protein called tau, which is normally found in conjunction with neuronal microtubules (a part of the cytoskeleton). In addition, one aspect of the neuropathy induced by annonacin is abnormal neuronal tau behavior. But a massively more frequent and consequential tauopathy is Alzheimer’s disease, so these findings raise the fascinating question as to whether environmental toxic agents might contribute to the burgeoning world-wide caseload of Alzheimer’s – and if so, how much, and under what genetic circumstances? The significance of such questions for public health in countries with increasingly ageing populations is obvious.
One point already alluded to above is the notion that a transient ‘hit and run’ exposure to a toxic substance might set up a continuous and actually self-perpetuating cycle of damage. Such a possibility could considerably complicate attempts to identify causative toxic agents. If a single short-live exposure (or transient set of exposures) to an agent can result in disease many years later, it is clear that fingering the original culprit becomes correspondingly more difficult. It remains a possibility that such effects are relevant to the cycad saga at least, but a more detailed consideration of this notion is a topic for a later post in this series.
In the meantime, a biopoly-verse rumination:
Bring genetics and host factors to view
Where some insidious poisons can brew
To stay and remain?
Or start off a chain
Of damage in an unfortunate few.
References & Details
(In order of citation, giving some key references where appropriate, but not an exhaustive coverage of the literature).
‘ A classic example in this regard is the insecticide DDT……’ (With respect to persistence in fat). See Turusov et al. 2002.
‘….arsenic (capable of replacing phosphorus) and thallium (capable of replacing potassium).’ With respect to arsenic, it is interesting to recall the recent controversy regarding ‘arsenical life’, where arsenic in a specific bacterium was reputedly replacing phosphorus (see a previous post for brief detail on this). Arsenic can compete with phosphorus when it is in the form of arsenate (See Kaur et al. 2011; and also Dani 2011 for a discussion of the biological significance of this). For more details regarding thallium and its competition with potassium, see Hoffman 2003.
‘….release of the metal component may simply liberate it for another cycle of inhibition. This can be overcome if a chemical agent (a chelator) is administered which is capable of tightly binding the metal, solubilizing it, and allowing it to be excreted. See Flora & Pachauri 2010; Jang & Hoffman 2011.
‘….potassium-40 (40K) …. has been proposed as a major source of natural mutation, although experimental results suggest that its contribution to mutation must indeed be subtle influence.’ See Gevertz et al. 1985 for more detail and a refutation of the importance of this radioisotope for mutation, at least in bacteria.
‘…..polonium-210 (210Po), …is present in tobacco smoke, and it has been attributed a major role in the generation of smoking-induced cancer….’ See Zagà et al. 2010.
‘ Polonium-210 has been in the news in recent years, through its use an exceedingly potent poison in the murder of the Russian Alexander Litvinenko…..’ Polonium-210 is an α-emitter (Helium-4 nuclei). While these emitted particles are relatively massive and poorly penetrating, they are very dangerous if an α-source has been ingested. Doses as little as 1 μg may be lethal in susceptible individuals, and doses of several hundred μg will be universally fatal. See Scott 2007. For more details on the Litvinenko case, see a BBC timeline article.
‘….polonium-210 can exert low-level effects if ingested in small enough doses.’ See also Scott 2007.
‘ The influence termed ‘cofactors’ ….. example is a putative requirement for the presence of simian virus 40 (SV40) for the generation of mesothelioma by asbestos….’ See Rivera et al. 2008; Qi et al. 2011. Note that SV40 was a contaminant of early Salk polio vaccine preparations (see Vilchez & Butel 2004).
‘….origins of Parkinson’s disease…..’ This disease (the ‘shaking palsy’) was first described in the early 19th century by Dr. James Parkinson (Thomas & Beale 2007), who thus bequeathed his name to it. Although obviously an eponymous title, the “Parkinson” is often now rendered with a lower-case ‘P’.
‘ These neurons are also pigmented…..’ Melanocytes, the cells in the skin which produce the pigment melanin responsible for skin color (along with the related pigment pheomelanin) are derived from the same embryological origins as neurons, the neural crest.
‘….a type of melanin (‘neuromelanin’)….’ Neuromelanin is chemically similar, but not identical to, the black melanocyte pigment, which itself is often termed ‘eumelanin’. See Zecca et al. 2001.
‘…..the source of the problem [Parkinson-like illness] was tracked down…..’ See Langston et al. 1983.
‘….widely reported in the scientific literature….’ For example, see an article in 1984 by Roger Lewin in Science, whose title (‘Trail of Ironies to Parkinson’s Disease’) speaks for itself.
‘…even found their way into popular fiction quite quickly….’ The well-known ‘new wave’ science fiction novel Neuromancer by William Gibson (Ace Science Fiction, 1984) features a particular scene where an individual is deliberately victimized by means of the nasty aspects of MPTP neurotoxicity. Since the book was first published in 1984, this was at the time a very quick uptake on a scientific and medical development.
‘ Those unmistakably victimized by MPTP had varying fates…..’ See Langston’s popular book (co-authored with Jon Palfreman), The Case of the Frozen Addicts (Pantheon, 1995). Also see a Wired magazine article.
‘…..a relative latecomer in 1817…..’ See the above note about James Parkinson.
‘….‘natural’ PD …. a toxic condition…?’ See Calne & Langston 1983.
‘….exposure to insecticides ….as a potential agent of PD …. not been firmly nailed down…’ See Brown et al. 2006.
‘ Most cases of sporadic PD occur later in life….’ Only 1-3% of total PD cases can be attributable to direct genetic causes (See Lorinicz 2006).
‘….MPTP itself is acted upon by a specific enzyme with the brain, monoamine oxidase….’ See Herraiz 2011 (a).
‘…..inhibitors of MAO enzymes are protective against the effects of MPTP…..’ Herraiz 2011 (b).
‘….also explains the high selectivity of MPTP (the precursor to MPP+) in its toxic action…’ For an early report on MPP+ uptake, see Javitch et al. 1985.
‘….it [MPP+] acts as a primary toxic agent towards mitochondria….’ For a little more detail on mitochondrial activity, see a previous post. For more on Complex I in general, and with respect to MPTP / MPP+, see Schapira 2010.
‘….epidemiologists have noted an unusual incidence ….ALS-PDC…’ For an entertaining account of the history of this topic, see The Island of the Colour-blind (Picador, 1996; Book Two, Cycad Island) by the famous neurologist Oliver Sacks. For a general overview of ALS-PD, see Steele 2005.
‘ Cycad flour fed to experimental animals…..’ See Shen et al. 2010.
‘ A specific amino acid …BMAA….has been repeatedly linked with cycad-induced disease…’ For a review and disputation of this, see Snyder & Marler 2011.
‘ Another contender is methylazoxymethanol….’ See Kisby et al. 2011.
‘….a specific compound from this fruit (annonacin) has implicated….’ See Champy et al. 2004; Lannuzel et al. 2008. Other compounds chemically related to annonacin have also been implicated: See Alvarez Colom et al. 2009.
‘…one aspect of the neuropathy induced by annonacin is abnormal neuronal tau behavior…’ See Escobar-Khondiker et al. 2007.
Next Post: This is the last post for 2011; will be back early next year.
From time to time, it will be appropriate to offer updates (or upgrades) of previous posts when it seems appropriate. In late March, I looked at ‘paradigm shifts’ in biological science, particularly in the context of so-called biological ‘dark matter’. Here a Table was provided with a list of some developments in recent bio-history which could qualify as paradigm shifts, especially against the current background where the meaning of a scientific ‘paradigm’ has been diluted in much of the literature. While this Table was not originally intended to be completely comprehensive, after the fact I have noted that a particularly important case was inadvertently overlooked. That is the subject of the current post.
The Chemiosmotic Hypothesis
Cellular processes require energy, and a universal energy ‘currency’ is the molecule adenosine triphosphate (ATP). It has been long recognized that the hydrolysis of ATP to the corresponding diphosphate (ADP) provides the free energy for driving a host of biological reactions. The synthesis of ATP itself is therefore of crucial significance, and naturally requires an energy source in order for this to be accomplished.
In 1961, a British biochemist by the name of Peter Mitchell published a paper in Nature outlining a novel proposal for the mechanism of the generation of ATP through the electrochemical properties established in certain biological membranes. These are found in prokaryotes, and also eukaryotes via their mitochondria (the ubiquitous organelles concerned with energy production) or chloroplasts (the plant cellular organelles mediating photosynthesis). Mitchell’s ‘chemi-osmotic’ hypothesis postulated that, rather than relying on an energy-rich chemical intermediary, oxidative phosphorylation (the synthesis of ATP from ADP occurring during respiration) was dependent on proton (hydrogen ion) flow across membranes. In essence, respiratory processes pump protons across an enclosed membrane boundary such that an electrical potential is generated across the membrane. Mitchell termed the ‘pull’ of protons back across the membrane as the ‘proton motive force’, or a proton current. This flow of protons could be directed through protein-mediated channels for the purposes of performing useful work.
Although now enshrined within the modern biochemical world-view, in the early 1960s this notion was quite radical, and not at all in tune with many of the ideas of most major researchers in the field at that time. In fact, it took over a decade a half before enough evidence was garnered to convince most remaining doubters. But Mitchell certainly had the last laugh, being awarded a Nobel Prize for his innovative proposal in 1978.
ATP Synthase and the Chemiosmotic Hypothesis
A remarkable catalytic complex at the core of ATP generation, the membrane-associated ATP synthase (ATPase), has had a central role in the ultimate acceptance of the chemiosmotic hypothesis. This resulted from studies on purified components of the synthase complex and reconstitution experiments, where directed proton flow across sealed model membranes (liposomes) was shown to be crucial for ATPase activity. In some ingenious experiments, the required proton flow was produced by the introduction of a protein involved with prokaryotic photosynthesis (bacteriorhodopsin) as a light-driven proton pump. (Other proton pumps from diverse biochemical sources could also perform similar roles). Such findings were subsequently reinforced by numerous structural and functional studies.
The ATPase has been revealed as a molecular motor driven by proton flow directed through the transmembrane (‘Fo’) component of the catalytic complex. The proton current is harnessed to provide energy for driving the physical rotation of the soluble (‘F1’) ATPase component, resulting in ATP synthesis at three catalytic sites. In some amazing cases of experimental virtuosity, this molecular rotation has been visualized in real time using fluorescent tags, and the association of rotation with ATP synthesis demonstrated by magnetic bead attachment to the F1 subunit, followed by artificial rotation induced by appropriate magnets.
The striking nature of the membrane-associated ATPase as a rotary molecular motor has inspired many offshoot thoughts and speculations. As a demonstration of a ‘natural nanomotor’, it would come as no surprise to hear that that the nascent field of nanotechnology has paid particular notice.
Why a Paradigm Shift?
So, it might be immediately seen that the proposal, experimental testing, and ultimate support for the chemiosmotic hypothesis is of great scientific significance, but is it really meaningful to refer to it as a paradigm shift? Well, yes, it is. Firstly, the initial resistance to this idea in itself is consistent with the view of shift in a paradigm requiring the upheaval and dismantling of an earlier view – if not by the death of an aging cadre of reactionary biologists, at least via their eventual accession to the concept through the accumulated weight of evidence.
But perhaps the most fundamental novelty of Mitchell’s ideas came from the inherent aspect of spatial organization of cellular structures in determining function, as he explicitly stated. In his own words, from his 1961 Nature paper:
“the driving force on a given chemical reaction can be due to the spatially directed channelling of the diffusion of a chemical component or group along a pathway specified in space by the physical organization of the system”.
In other words, structures on a cellular scale (membranes, in this case) can serve as a basis for directing biochemical reactions in specific ways, and this general effect has also been termed ‘vectorial biochemistry’. This view was a radical proposal in the early 1960s – and accordingly met with considerable resistance. In fact, cells are not just ‘bags of enzymes’, but partitioned in complex ways into different compartments, and this partitioning is very significant for specific functioning. This is particularly so (as we have seen) for bioenergetics.
The development of some form of membrane compartmentalization of proto-cells during the early stages of the origin of life is recognized as a major evolutionary transition. Its importance can be inferred from simple logic, since an evolving molecular biosystem could never undergo progressive selection and functional advancement were its components not restricted into a bounded spatial compartment. Dilution of reactants would otherwise rapidly remove any useful molecular innovations, and bring in potentially interfering molecules. Included among the latter are likely parasitic systems, whose unchecked activities would be a permanent stumbling block. But the long-term implications of the chemiosmotic principle show us that biological membranes are much more than just phospholipid sacks demarcating collections of biological molecules from the external environment. They are integral and essential parts of biological operations in their own right. And their evolution into these roles is a very ancient event in the history of life. Leaping from early biogenesis to future human aspirations, the importance of membranes and higher-level structures for vectorial direction of function should not be forgotten when artificial cell design is contemplated.
So Mitchell’s contribution is duly inserted into the original ‘paradigm shift’ Table thus:
It is also notable that this year marks the 50th anniversary of the publication of Mitchell’s seminal paper.
And finally, a biopoly(verse) salute to the pioneer:
The hypothesis chemiosmotic
Made Mitchell seem quirky and quixotic
But opinions revise,
And then a Nobel Prize
Sealed the field as no longer exotic.
References & Details
‘……a British biochemist by the name of Peter Mitchell published a paper in Nature…’ See Mitchell 1961.
‘….Mitchell ….. awarded a Nobel Prize for his innovative proposal in 1978.’ See Harold 1978; also the Nobel organization site for the 1978 Chemistry prize. See also a relevant piece in Larry Moran’s Sandwalk blog. Mitchell died in 1992.
‘…..studies on purified components of the synthase complex…..’. A major contributor to these studies was Efraim Racker (1913-1991), A biographical memoir by Gottfried Schatz (National Academies Press, online) provides an excellent background to this and numerous related areas. Paul Boyer and John Walker also were pivotal in structure-function studies regarding ATP synthase, for which they received the Nobel Prize for Chemistry in 1997. For a very recent and comprehensive review of the membrane-associated rotary ATPase family, see Muench et al. 2011.
‘…..the introduction of a protein involved with prokaryotic photosynthesis….’ See Racker et al. 1975.
‘…..nanotechnology has paid particular notice….’ See Block 1997 (Article title “Real Engines of Creation”, which refers to K. Erik Drexler’s book Engines of Creation, a pioneering manifesto of the potential for nanotechnology – Doubleday, 1986). Also see Knoblauch & Peters 2004.
‘…..artificial cell design…..’ See a previous post on synthetic genomes and cells for more on this cutting-edge topic.
Next Post: Regrettably, work commitments enforce a temporary hiatus on biopolyverse posts until early December. But will return then!!
A considerable number of the recent series of posts have been concerned with molecules that can be referred to as drugs. It seems useful here to take a look at this from a semantic point of view.
Drugs at Different Levels
Most people carry around in their minds more than one specific meaning ascribed to the small word ‘drug’. If you hear “He’s a drug dealer” or, “She’s on drugs”, the references are not likely to be to antibiotics or blood-pressure medication. Conversely, the sentence, “My doctor prescribed a drug for me” is most unlikely to refer to crystal meth. As a result, the great majority of people realize (even if only intuitively) that the illegal band of drugs are but a subset of a much larger group, that includes substances both universally approved and potentially truly life-saving. It would then be reasonable to assume the definition of a ‘drug’ in this larger sense should be fairly straightforward.
Some standard dictionary references are along the lines of: “A substance used in the diagnosis, treatment, or prevention of a disease or as a component of a medication” or, “a chemical substance that affects the processes of the mind or body”. The US Food & Drug Administration (FDA) defines drugs with wording introduced by the Food, Drug, & Cosmetic Act of 1938, as “articles (other than food) intended to affect the structure or any function of the body of man or other animals” [Sec. 201(g)(1)]. These definitions are quite broad, especially the latter. But speak of ‘drugs’ with a pharmacologist, and small molecules are most likely to be the topic of conversation, and not just any small molecules. Indeed, the term ‘drug-like’ is frequently used in the general field of drug discovery to encapsulate (so to speak) the special features which a successful medicinal drug should embody. Obviously, a drug must have definable function(s), which means that it must be directed to a specific molecular target or a limited set of targets (very often, but by no means exclusively, proteins). But a number of additional properties are very important if the drug is to successfully survive in an active form long enough to be useful, and to find its way to the desired target when administered to a patient. For example, a simple set of guidelines for evaluating a candidate compound formulated by Lipinski has been termed the ‘rule of five’, owing to the recurrence of five (or multiples of it) in the definition of the useful range of properties to look for. Getting a drug to where it needs to go is the preoccupation of the burgeoning field of drug delivery, which now intersects in many cases with advances in nanotechnology.
These rules were designed expressly with reference to small molecules, since increasing molecular size is often associated with diminishing returns in terms of delivery, and sometimes physical properties such as solubility. But that is certainly not to say that large molecules cannot be useful pharmacological and therapeutic agents. In the previous post, it was noted that it has only been in very recent times (historically speaking) that large proteins (especially antibodies) could be garnered from the biosphere for useful human applications. In fact, (as also noted), antibodies have become a billion-dollar industry, especially where monoclonal or specifically engineered antibody variants are concerned. And these antibodies are often referred to as drugs, especially in lay usage. Though obviously moving beyond a tight pharmaceutical definition of ‘drug-likeness’, this is perfectly consistent with the above broad definitions, including that from the FDA. But there are some gray areas…..
Drugs and Category Overlap
It is quite clear that some classes of substances or preparations may have members which have dual drug and nondrug characteristics. One such case noted by the FDA is the field of cosmetics, where even more narrow types of products differ in this kind of duality. For example, shampoos may be seen as primarily hair cleansing, or cosmetic, preparations, and indeed many are simply that and no more. But certainly some have additional functions, such as treating fungal (dandruff) or louse infestations. In this case, specific compounds are added for the indicated medicinal specifications. Although it is obviously of practical significance for product safety and efficacy requirements that some preparations should be regulated and licensed as both drugs and cosmetics, here the relevant products are mixtures, and specific molecules are not doing functional double-duty. For example, an anti-dandruff shampoo may have many different components, but the most significant are the detergent (usually sodium lauryl sulfate, for the cosmetic washing function) and the dandruff inhibitor (specific compounds such as zinc pyrithione). In other words, we cannot speak of either of these individual compounds as having an overlapping drug / cosmetic function; it is only as mixtures (along with various other materials) that the product as a whole acquires this status.
Yet there are certainly well-defined cases where specific molecules share categorization as a drug in combination with other properties. A prime case in point is a contender for the title of oldest drug used by humans: ethyl alcohol, or ethanol. Its psychoactive and other physical effects clearly indicate its drug status, but ethanol can also be metabolized to yield specific calorific value. As such, it is then a food as well as a drug. This is straightforward, but some other areas of ‘foods’ are less so. One definition of a ‘food’ might focus on the ability of a substance to be digestible, or act as source of energy, but clearly this is not sufficient for a healthy diet. There are numerous nutritional ‘cofactors’ which are essential for human health, included among which are a number of inorganic elements (principally metals, but also some other trace elements), and a group of vitamins.
Where do vitamins stand with respect to drug classification schemes? As small molecules which act as organic enzymatic cofactors for catalysis (coenzymes), the defined vitamins are an essential human dietary requirement, owing to our inability to synthesize them. But since they are not directly digestible themselves, they are classified by the FDA as ‘dietary supplements’, which are encompassed within the broader area of foods, and not drugs. Vitamins, then, fall into the this category and therefore would escape labeling as drug materials, unless they were chemically altered from the natural forms. Most dictionary definitions of ‘food’ also include vitamins. Even so, in other quarters vitamins have been clearly depicted as drugs. One basis for doing so is that vitamins can clearly cure diseases – but since the relevant diseases are deficiencies of the vitamins themselves, this would seem to be a special case. As we have seen with alcohol, assignment of a specific compound as a food does not mean it cannot also be a drug. But clearly there is a difference here: vitamins are essential for life, while alcohol (whatever some people might say) is not.
The figure below depicts two separate classifications where the vitamins are considered either as drugs (A) or not (B):
Two depictions of drug categorization and its overlapping areas. These are not to scale in terms of the relative sizes of the respective groups, and are schematic only. ‘Food / nutrients’: This refers to subtances which directly provide energy, structural building blocks, or essential assistance with normal metabolic functioning. ‘Dietary cofactors’ in general include both vitamins and inorganic substances (such as essential metals). ‘Proteins / macromolecules’: Not all macromolecular therapeutic agents are proteins, as for example nucleic acid aptamers. A, Vitamins considered as drugs. Some cosmetic preparations contain vitamins, so vitamins are shown to intersect with the ‘drug-overlap’ region of all cosmetics. B, Vitamins excluded from classification as drugs.
Does this cover everything? Well, there is an additional broad grouping of substances termed ‘nutraceuticals’, a hybrid term from ‘nutrient’ and ‘pharmaceuticals’. A nutraceutical in principle can be any food source component with biological properties outside of direct nutrition, but many of the best-known examples are antioxidants. Included among these are phytoestrogen compounds, considered in an earlier post. Resveratrol in particular (see the relevant Figure from this same post ) has generated enormous interest for its observed anti-ageing effects.
Where do these compounds reside in the above figure? Although they are by definition associated with some kinds of foodstuffs, they are neither directly digested (as for proteins, and digestible carbohydrates and fats), nor required for essential metabolism (as for vitamins). Therefore, it is logical that they be considered a subset of the large drug category, outside of the macromolecular subregion, as shown above. These compounds can be identified, purified, synthesized, and administered independently of their original sources. In this respect, they are no different from any other small molecule natural products derived from the biosphere.
Drugs as Foreign?
Can drugs be thought of as molecules which are ‘foreign’ to the body to which they are administered? (In other words, compounds which are not synthesized by the human or animal which receives them). In a strict sense, this would include vitamins which are dietary essentials through the lack of synthetic machinery for their production by a host animal or person. But there are problems with this proposal, and vitamins themselves are a case in point. For example, although Vitamin C is essential for human health (scurvy resulting in its absence), rats, mice and numerous other species have no problem making their own. Is Vitamin C then a drug for humans (capable of curing scurvy) but not for rats?
And numerous human proteins can be administered under circumstances where they can be considered drugs. Antibodies are an interesting case in point. Originally, monoclonal antibodies were of murine origin owing to the technological requirements of their production. The xenogeneic (foreign) nature of these proteins resulted in the induction of immune responses against the monoclonal antibodies themselves, when they were given to patients. In more recent times, fully human monoclonal antibodies have been developed, in order to circumvent this very significant problem. Yet an antibody of this type is still not literally and totally ‘self’, since its specific combining site is generated by recombinational and mutational mechanisms such that its exact sequence is not directly encoded in the human germline.
But non-variable molecules both large and small also come into this picture. Think of human growth hormone, of value for treating some forms of dwarfism – and sometimes abused for the purposes of body-building. Numerous other proteins and small molecule hormones can also be cited – so the notion of ‘foreign-ness’ for drugs in general becomes untenable.
Drugs, Poisons, and Doses
Drugs have been termed ‘poisons that save lives’, which carries the implicit message of the importance of dosage. But stating that ‘all drugs are poisons’ may be technically correct at a broad enough level, yet not particularly useful, given the vast differences in dosage ranges for efficacy vs. safety seen with different therapeutic compounds. Here a balance or ‘window’ must be found between the two poles of beneficial drug activity and unacceptable toxicity. The old saying ‘the treatment was successful, but the patient died’ provides an ironic testament to this inherent dilemma of drug pharmacology.
As an example of the great range of drug therapeutic windows which can exist, consider the treatment of syphilis. The pioneer of chemotherapy, Paul Ehrlich, found an arsenical compound (Salvarsan) which became an effective treatment for syphilis through its activity against the bacterial causative agent Treponema pallidum. But its toxicity at therapeutic doses was a major problem encountered in a high percentage of patients, so it was clearly not ideal. When penicillin became available in the 1940s, it was not only highly effective but also associated with very low toxic side effects. Indeed, the rising problem of bacterial resistance was initially countered by simply increasing the dosage of penicillin (or its many derivatives) without problems – but of course this soon becomes ineffectual as resistance increases. (Penicillins can actually induce serious problems through allergies in a minority of people, but this is quite distinct from direct toxicity).
There is also a piece of folk-wisdom along the lines of ‘too much of anything can hurt you’, which is certainly true for some natural nutritional requirements as well as drugs. In a general sense, too much food is clearly bad through the development of obesity, but the ‘dosage’ effects of nutrients can be observed in a much more specific manner. We can look within the set of vitamins once more for useful comparisons, which also demonstrate similar variation in the ‘safety’ windows of dosage as seen within artificial drugs. Vitamin C has exceedingly low (if any) toxicity, for example, and some people have routinely taken very high doses of it for long periods as part of ‘megavitamin’ therapy. On the other hand, the fat-soluble Vitamins A and D are unquestionably highly toxic when taken in excess of recommended daily requirements.
The dosage effect can also be related to the above observation that drugs need not be alien to the biochemistry and physiology of the patient (or animal) undergoing treatment. A pathology caused by a deficiency in a specific molecule can be corrected through artificial intervention. Conversely, certain pathological states may benefit from the provision of ‘unnatural’ administration of normal bodily proteins, such that the circulating amounts of the factor of interest are maintained for therapeutic purposes at higher levels than would normally be the case.
After this short foray into some issues surrounding the meaning of drugs, I’ll conclude with references to ‘nutraceuticals’ once more, by means of a biopoly(verse) note.
By analyses really quite shrewd
On mixtures both complex and crude
Smart chemists have shown
(And now it is known)
Natural drugs exist in some food.
References & Details
‘….. (FDA) defines drugs….’ For FDA definitions of both drugs and cosmetics, see the relevant page of the FDA site.
‘….set of guidelines for evaluating a candidate compound formulated by Lipinski…’ For a discussion of the basis of the Rule-of-five and moving beyond it, see Zhang & Wilkinson 2007.
‘…..field of drug delivery, which now overlaps with advances in nanotechnology.’ For a recent review of this topic in the cancer field, see Chidambaram et al. 2011.
‘….sodium lauryl sulfate….’ Also known as sodium dodecyl sulfate, this detergent also has wide application in laboratories as well as cosmetics.
‘…..a contender for the title of the oldest drug used by humans….’ Often alcohol is definitively cited as the oldest drug. I call it ‘a contender’ here since (as noted in an earlier post), the use of botanical medicines is also very old, and can even be linked with primate behavior (see a previous post on zoopharmacognosy). On the other hand, the use of alcohol (or abuse, depending on one’s views) is possible simply from natural cases of fruit fermentation, also seen with animals. So the origins of alcohol sampling by humans need not require any technology, and is undoubtedly of great antiquity.
‘……ethanol can also be metabolized……’ For an example of the differing influences of food vs. drug effects in an animal system, see Dole et al. 1985.
‘…..classified by the FDA as ‘dietary supplements’….’ For the FDA definitions of dietary supplements, see the relevant page of the FDA site.
‘…..vitamins can clearly cure diseases…..’ This was noted by Tulp et al. 2006, and that vitamins were thus ‘drugs by any definition’. Taken literally, this is clearly incorrect (one can simply exclude vitamins from a drug definition as dietary supplements, as for the FDA).
‘…..nucleic acid aptamers….’ (Figure footnotes). See a previous post for a brief consideration of RNA aptamers. From large libraries of variants, RNA molecules can be selected to bind desired ligands, and this can be used therapeutically. The first therapeutic aptamer (‘pegaptanib’) was directed against a specific form of vascular endothelial growth factor, for the treatment of ocular vascular diseases. See Ng & Adamis 2006.
‘……an additional broad grouping of substances termed ‘nutraceuticals’……’ For example, see Tulp et al. 2006.
‘……Resveratrol in particular …… has generated enormous interest for its observed anti-aging effects……’ See Pezzuto 2011.
‘……Vitamin C is essential for human health (scurvy resulting in its absence), rats, mice and numerous other species have no problem making their own.’ See Martí et al. 2009 for more information. The production of Vitamin C from glucose requires the enzyme L-gulonolactone oxidase, which humans, primates, and guinea pigs lack. Lachapelle & Drouin 2011 look at when this occurred in evolutionary time.
‘…..its exact sequence is not directly encoded in the human germline.’ Antibodies are composed of constant and variable regions, where the variation of the latter accounts for the vast range of different antibody binding specificities which can be induced by immunization. Particular variable region sequences allowing antigen recognition are specific to that immunoglobulin molecule, and are termed an ‘idiotype’. See Searching for Molecular Solutions Chapter 7 for a more detailed discussion of this.
‘ Numerous other proteins and small molecule hormones can also be noted…..’ Proteins such as interferons were noted in the previous post. Small molecules include adrenalin, thyroid hormones, and natural steroids. In all such cases, though, there is the potential (realized in many cases) for rendering such molecules ‘non-natural’ by various forms of artificial tinkering to improve their performances as drugs.
‘….pioneer of chemotherapy, Paul Ehrlich…’ See Thorburn 1983 for some biographical and other relevant information.
‘……certain pathological states may benefit from the provision of ‘unnatural’ administration of normal bodily proteins…..’ Again, see the reference to the example of interferons in the previous post.
‘ Vitamin C ….. ‘megavitamin’ therapy.’ The Nobel Prize-winning chemist Linus Pauling was a notable proponent for the efficacy of large Vitamin C (ascorbate) doses for conditions ranging from viral infections to cancer. (For example, see Pauling & Moertel 1986). However, no experimental evidence has validated these claims.
‘…..fat-soluble Vitamins A and D are unquestionably highly toxic….’ It is notable that the livers of certain polar animals (including bears and seals) are very rich in Vitamin A, and the eating of such livers by polar explorers has resulted in Vitamin A poisoning (hypervitaminosis A). See Rodahl & Moore 1943.
Next Post: Two Weeks from now.
It was noted in the previous post that most natural molecules of use to humans (‘chrestomolecules’ ) have traditionally been of low molecular weight, with the exception of simple biopolymers. Here it is time to have a brief look at the scope of large molecules and even larger biological systems which can be sampled from the biosphere and usefully applied to human activities.
Big Uses for Big Molecules….
Large biomolecules such as proteins can obviously act as nutrients, but this special category of ‘usefulness’ is not the focus of interest here. Indeed, in this context it should be emphasized that the benefits of consuming a protein as a foodstuff for any organism (humans included) result from the acquisition of its amino acid building blocks. ‘Digestion’ is intimately associated with breaking down consumed nutrients into components which an organism can employ for its own biosynthetic requirements. The original three-dimensional structure and function of the consumed protein is clearly irrelevant for such a nutritive purpose. But when we are considering the utility of large biomolecules for other human needs, the protein shape and function is crucial.
Biotechnological developments in the past century or so have extended the field of molecular utility to include a diversity of proteins (noted in part of in the ‘chrestomolecule’ Table of a previous post). The great majority of these can be grouped into the functional categories of natural biocatalysts (enzymes) and natural specific binding molecules (antibodies), but some additional cases can be noted:
Let’s take a quick look at some specific examples among these large protein sets:
Looking for Enzymes in All the Right Places
Many enzymes from a variety of biological sources are used in industrial processes, and several examples are of particular interest from a molecular biological point of view. There is at this time considerable interest in biological resources obtainable from ‘extremophile’ micro-organisms, or literally ‘lovers of extreme environments’. Certain bacteria thrive under amazing extremes of temperature, salinity, pH or pressure, and bacterial biosystems mediating metabolic processes and growth must in consequence be selected for tolerance of each type of extreme conditions. Only in quite recent times has it become recognized that the earth harbors an entire thermoresistant prokaryotic ecosystem which lives by chemical energy independent of solar radiation. These organisms may be found in geothermal hot springs (as in Iceland or Yellowstone National Park), at deep underground sites or at thermal vents in the deep oceans.
Thermophilic (heat-stable) enzymes in general have many applications, but probably the most famous thermophile application in molecular biology is the use of the DNA polymerase from the hot-spring bacterium Thermus aquaticus, or Taq polymerase. (DNA polymerases are of fundamental importance for the copying and replicating of an organism’s DNA during cellular growth). The hardy Taq polymerase has made the polymerase chain reaction (PCR) a practical proposition, which has had an enormous impact in all areas of this and related sciences and gained its originator (Kary Mullis) a Nobel Prize. PCR involves replication and amplification of a DNA duplex in the laboratory through successive cycles of strand denaturation, association on each strand of specific primer oligonucleotides (short DNA segments), and primer extension (strand replication) with the DNA polymerase. Since strand denaturation is achieved by heating to 95˚ C or greater, a conventional polymerase present in the reaction is ‘killed’ and must be (very tediously) re-added for each cycle. A thermostable DNA polymerase, however, can be maintained throughout the course of the reaction. Although many other such heat-resistant polymerases are compatible with PCR, Taq polymerase is still the most widely used. At the other extreme from thermophilic organisms are psychrophiles, which thrive at very low polar temperatures, and are also potential sources of novel enzymes for novel applications.
In the ‘chrestomolecule’ Table of a previous post, antibodies (immunoglobulins) were featured. These important effector molecules of the mammalian adaptive immune system can be regarded as natural binding molecules produced ‘to order’ in response to specific stimuli (usually threats from invading external organisms), by diversification and selection mechanisms. Their applications are many, but can be divided into in vivo tasks which relect the original biological role for antibodies, or in vitro roles. Even naturally-formed antibodies within an individual are obviously useful if they mediate recovery from microbial infections, and the production of antibodies within a host has long been artificially directed by means of vaccines. Relatively recently compared to the first use of vaccination (attributable to Edward Jenner and cowpox immunization against smallpox), passive immunity has emerged as a therapeutic tool. This can be effected by transfer of whole serum or serum components rich in immunoglobulins from immune animals or humans to non-immune recipients, for the purposes of their protection from sudden exposure to a toxic or infectious environmental agent. With passive immunization even with crude serum fractions, antibodies qualify as examples of large natural ‘chrestomolecules’. But in modern times, passive immunization goes far beyond this.
This essentially began with the discovery of monoclonal antibodies in 1975, and has led to the vast field of antibody engineering. The manipulation of antibodies has wide therapeutic applications, and products based on antibodies have become a billion-dollar earner for the biotechnology industry. But engineered (and conventional) antibodies have many uses in vitro as well, as noted above. These include diagnostics and sensors, and for a variety of purification purposes in research and industry. In its range, this topic really requires further attention in a later post. But before moving on, it’s worth noting that the fields of antibodies and enzymes are not mutually exclusive. For a couple of decades, it has been known that antibodies can be raised such that they catalyze specific chemical reactions. This is possible through clever immunization strategies, where host animals are immunized with analogs of chemical transition-state intermediates of enzymatic catalysis. Even so, the best artificially-generated catalytic antibodies fall short of the efficiencies observed with natural protein enzymes.
And Other Proteins….
Although antibodies remain the star players, the adaptive immune system can contribute other useful large molecules. The key adaptive receptor of the cellular arm of the vertebrate immune system is the T cell receptor, signaling through which activates various different classes of effector T cells. Engineered T cell receptors in a number of forms have application in some cutting-edge technologies for human therapies. Some other proteins which modulate the functions of immune and other cells also have therapeutic and commercial significance. Included among these are interferons, known initially for their antiviral properties, but also for their immunomodulatory activities.
Beyond the immune system, various other proteins are mostly important through their contributions to knowledge, which have enabled the subsequent design of other useful structures. For example, the zinc-finger protein motif mediating DNA-binding has been intensively studied, allowing the derivation of a ‘code’ for binding and the generation of artificial binding motifs towards virtually any desired DNA sequence of 18 base pairs. When teamed with a nuclease domain, such tailored ‘zinc finger nucleases’ have tremendous potential in a number of clinical contexts. In different areas, numerous proteins have contributed their structural frameworks themselves for useful applications. This principle has been used for development of protein frameworks which can replace immunoglobulins for the specific recognition of desired binding targets.
….and even Bigger Higher-Level Useful Systems
By analogy with the introduced term ‘chrestomolecule’, an economically useful biological system (involving numerous mutually interactive molecules) could be termed a ‘chrestosystem’. But if we introduce biosystems into this picture, there are many levels of complexity that might apply. Does a whole organism thus qualify? Is even a domestic mammal a high-level ‘chrestosystem’? (After all, such animals have had many uses over the millennia in addition to being raised as food sources). And downward from there, certain fungi, yeast, and bacteria have long had uses in the modification of foodstuffs, long before they were even recognized as living organisms.
Perhaps cows (and so on) might indeed be viewed as simply highly complex organized systems in the service of humanity, but for the present purposes it will be most constructive to restrict our focus onto viruses. Viruses are usually considered as life-forms, and often have sophisticated regulatory controls, but are nevertheless many orders of magnitude less complex than a large multicellular organism. In their simplest forms, they can be regarded as small nucleic acid genomes packaged within a protein coat, and thus have a relatively small set of macromolecules which collectively allow them to infect their target cells and reproduce. So it is worth considering how some viral systems have useful properties for artificial exploitation. These will be grouped into bacterial viruses (bacteriophages, often referred to simply as ‘phage’) and a select group of animal viruses:
The ‘other viral’ categories are relevant examples, and not intended to be exhaustively comprehensive. (Many other cases exist).
Phage For This Age
Viral parasites of bacteria and other prokaryotic organisms, phage in general are extremely diverse in structure and genetic organization. Phage must attach to their specific prokaryotic host and ensure that their genomes (single or double-stranded DNA or RNA) enter the host cell. Usually the result of this is viral hijacking of the host and its conversion into a phage factory, ultimately leading to host cell destruction (lysis) and liberation of a large number of new phage particles. In broad outline, or course, this is a universal propagation strategy used by viral parasites across all kingdoms of life. Sometimes phage can be carried by their hosts indefinitely by a process termed lysogeny, where the phage integrates into the host chromosome or is carried as an independent replicating genome (plasmid replicon). Study of these and other features of phage biology has yielded a wealth of productive information. Owing to the relative simplicity of phage genomes, it is in fact no exaggeration to state that the rise of molecular biology itself derives largely from phage studies.
Phage have been major contributors to numerous biotechnological advances, and providers of information of general molecular biological significance. These viruses of prokaryotes can also be used as specific information sources potentially leading to novel therapeutics. In the case of pathogenic bacteria, systematic study of their phage has been shown to be a useful approach towards identifying phage-expressed proteins or peptides directed against bacterial host cell components. Identification of both the phage products and their host targets allows screening systems to be established for finding low molecular weight chemical inhibitors of the host cell targets themselves. Owing to the specificity of phage interactions with bacterial hosts, drug-modified phage have also been considered as vehicles for drug delivery to desired target cells. Modified phage have also been tested as vectors for transfection of mammalian cells with protein or nucleic acid ‘payloads’.
The use of bacteriophage in the never-ending war against bacterial pathogens does not stop with phage protein or nucleic acid components. Perhaps inspired by the dictum, ‘the enemy of my enemy is my friend’, phage themselves have long been considered as therapies for a variety of bacterial diseases. The idea is simple enough: kill infecting bacteria with their own parasites. Since phage are specific to their bacterial hosts and cannot replicate in eukaryotes, safety also is not likely to be a problem. Yet the history of ‘phage therapy’ has seen conflicting claims of efficacy, although many successful treatments have been reported. In recent times there has been renewed interest in the potential of phage therapy, accompanied by the rise in antibiotic-resistant bacteria and a slow-down in the pipeline of new replacement drugs. The great diversity of phage has been suggested to be superior to the diversity of natural antibiotics themselves. The unique feature of phage used in this manner is that they are self-replicating, indeed the first therapeutic agents in this category. Their capacity for self-replication in turn is directly linked with the population of their target hosts, which gives the pharmacokinetics of phage therapy some unusual aspects. It has been suggested that inadequate understanding of these novel pharmacokinetics is a major source of the mixed results previously seen with trials of phage therapy, and due attention to this detail may well shift phage therapy into a mainstream alternative to antibiotics. This is especially likely in cases of bacterial infections which are resistant to the current antibiotic armamentarium. Apart from direct therapeutic applications, phage have also been considered for environmental use in the reduction of bacterial loads in human-derived wastewater.
Another interesting feature of the exploitation of phage as whole viruses is that they are not a one-molecule ‘magic bullet’, and are not categorizable as ‘molecular solutions’ in a the sense of involving a single molecular entity. (Certainly a phage could not be deemed a ‘chrestomolecule’, although its subunit molecules could if they were of value in isolation). Phage often use elegant genetic systems for their regulation, and also show highly efficient and compact biological organization. (For example, the phage ΦX174 provided the first natural demonstration of overlapping genes). But despite such layers of underlying organizational complexity, phage have simplicity at the level of the types of their molecular constituents, all consisting of their informational nucleic acids (genomes) protected by a protein coat (capsid), combined with an entry system into their specific hosts. It then follows that the use of phage (whether natural or engineered) as therapeutic agents is an early example of exploitation of a (replicating) supramolecular system.
A huge abundance of as-yet uncharacterized phage are still to be found in the natural environment, and if past experience is anything to go by, such phage will provide rich fields of novel biodiversity. In an extension of the earlier overview (in the previous post) of the bounty of marine bioproducts, marine bacteriophages have been cited as the largest reserve of untapped genomic information. In support of this contention is the observation that such phage have a very high proportion of unassignable open reading frames (predicted proteins which cannot be recognized as belonging to current known protein families). Current high-throughput sequencing technologies not only rapidly reveal new phage genes but allow high-altitude phage genomic comparisons, which are greatly facilitating studies of phage origins and evolutionary processes.
Other Viruses, or: What’s HIV Ever Done For Us?
Many viruses afflicting humans are infamous for the terrible toll they take on human lives, and for the resulting human suffering. Yet certain features of animal viruses can be harnessed towards desirable ends, especially their proficiency for targeting and invading specific host cells. As weapons against cancer, specific viruses have been modified and used to promote beneficial immune responses against cancers or infectious agents. Alphaviruses (a viral family which includes human encephalitis viruses) have been used as vectors to promote directed anti-tumor immune responses. Alternatively, viruses can be designed to directly kill (lyse) tumor targets, with viral examples including herpesviruses and poxviruses. (Obviously, in all such cases, it is of great importance that the introduced genetic modifications inactivate the ability of the virus to damage normal cells).
Even the human immunodeficiency virus (HIV), the causative agent of AIDS, has had something to offer. As a member of the lentivirus family, HIV has the useful property of infecting non-dividing cells, which many other viruses cannot accomplish. Vectors have been derived from ‘gutted’ versions of the HIV genome which are unable to complete the usual replication cycle, but retain the ability to insert a copy of the viral RNA as a DNA sequence in the host genome. These features render them an attractive means for transferring desirable gene sequences to recipient cells of interest. For example, such lentiviral vectors have been used to transfer specific T cell receptors to T cells from human cancer patients. In such cases, the novel engineered T cell receptors are designed to recognize tumor-specific antigens, and allow the transduced T cells to then recognize and kill tumor targets. The lentiviral vector-mediated gene transfer process is also useful for gene therapy applications.
It is interesting that a pathogen as serious as HIV, the destroyer of tens of millions of human lives since its full-scale emergence in the early 1980s, can ‘give something back’ by way of contributing a useful system for human application. Not that this would compensate its victims, of course, but the very fact that anything positive can be gleaned from the HIV experience says something about the human ability to recognize natural opportunities when they arise. There are many other examples of ‘positives’ accruing from dangerous viral and bacterial agents of disease, but these have only been made possible through technology. In many forms, modern biotechnology provides both the means to perceive useful biological opportunities (whether from pathogens or otherwise), and also pathways towards their beneficial application.
Homage to Nature, and then Moving Beyond It
In the molecular domain, Nature’s bounty is translatable into human benefit at several levels, as we have seen. There is firstly of course the direct discovery and application of low molecular weight bioproducts (detailed in the previous post), which has been done for thousands of years. The data provided by specific chrestobiomolecules can be used as a stepping-stone to even more effective agents, through successive rounds of human design intervention.
So, in the long run, the most important thing that large natural biomolecules and biosystems provide to us is information – information that acts as a spring-board towards the development of our own complex molecules and biosystems which are tailor-made towards specific applications. When this process is taken to its logical conclusion, we will reach a stage when we supersede the need to use such incredibly useful biological resources.
The use of higher-level systems in the future will thus inevitably become less a slavish derivation or copying of a natural precedent, and more and more an artificial improvement for specific design ends. This trend then increasingly overlaps with the field of synthetic biology, considered in several previous posts (30th May, 7th June, 20th June ).
Finally, a little comment on big molecules:
About natural products, one should recall:
They go far beyond molecules that are small
And think, if you please
That included in these
Are some considered the most useful of all.
References & Details
‘ Certain bacteria thrive under amazing extremes of temperature, salinity, pH or pressure…..’ See Van den Burg 2003.
‘…..earth harbors an entire thermoresistant prokaryotic ecosystem…….’ See Gold 1992.
‘ Thermophilic (heat-stable) enzymes in general have many applications….’ See Haki & Rakshit 2003.
‘….the polymerase chain reaction…’ For more details, see the accompanying ftp site for Searching for Molecular Solutions, for Chapter 4, Section No. 6 (‘PCR’).
‘….natural binding molecules produced ‘to order’ …….by diversification and selection mechanisms…..’ In brief, somatic diversification within immune cells of an vertebrate with an adaptive immune system results from combinatorial genomic rearrangements and somatic hypermutation, coupled with selection and amplification processes for cells uniquely expressing receptors recognizing a target antigen. This is also described (and referenced) in Chapter 3 of Searching for Molecular Solutions.
‘…..passive immunity has emerged as a therapeutic tool…..’ Among a vast number of examples can be found ‘antivenoms’ produced in horses or goats against various natural toxins from snakes, spiders, jellyfish, and other toxic organisms.
‘…..discovery of monoclonal antibodies…..’ See Köhler & Milstein 1975.
‘…….products based on antibodies have become a billion-dollar earner…..’ See Reichert et al. 2005.
‘……Engineered T cell receptors in a number of forms have application….’ More details (and references) and provided in the Cited Notes for Searching for Molecular Solutions in the accompanying ftp site, for Chapter 7, Section 18, T Cell Receptor Technology and Applications.
‘….interferons, known initially for their antiviral properties, but also for their immunomodulatory activities….’ Modified isoforms of interferon-alpha have been widely used (in conjunction with antiviral drugs) for treatment of Hepatitis C (See Tsubota et al. 2011); interferon-beta has been used as a therapy for the autoimmune disease multiple sclerosis (See Rudick & Goelz 2011).
‘…..development of protein frameworks……’ This is considered in Chapter 7 of Searching for Molecular Solutions.
‘……the rise of molecular biology itself derives largely from phage studies…..’ See Cairns, J., Stent, G. & Watson, J. Phage and the Origins of Molecular Biology. (Cold Spring Harbor Laboratory Press, 40th Anniversary Edition, 2007).
‘…….screening systems to be established for finding low molecular weight chemical inhibitors of the host cell targets themselves…..’ See Liu et al. 2004.
‘….drug-modified phage have also been considered as vehicles for drug delivery….’ See Yacoby et al. 2007.
‘……the history of ‘phage therapy’ has seen conflicting claims of efficacy…..’ See Sulakvelidze 2005.
‘……The great diversity of phage has been suggested to be superior to the diversity of natural antibiotics themselves…..’ See Schnaitman 2002.
‘…..the pharmacokinetics of phage therapy [has] some unusual aspects…….’; ‘……inadequate understanding of these novel pharmacokinetics is a major source of the mixed results….’ See Payne & Jansen 2003; Levin & Bull 2004.
‘…….phage have also been considered for environmental use…….’ See Withey et al. 2005.
‘…….the phage ΦX174 provided the first natural demonstration of overlapping genes…..’ When genes ‘overlap’, portions of their coding sequence occupy the same DNA tract, but in different reading frames. For the ΦX174 precedent, see Barrell et al. 1976.
‘…….marine bacteriophages have been cited as the largest reserve of untapped genomic information…….’ See Paul & Sullivan 2005.
‘……Alphaviruses …… have been used as vectors to promote directed anti-tumor immune responses.…..’ See Avogadri et al. 2010; ‘…..viruses can be designed to directly kill (lyse) tumor targets (including herpesviruses…..’ See Todo 2008; ‘……and poxviruses…..’ See Kirn & Thorn 2009.
‘…..many other examples of ‘positives’ accruing from dangerous viral and bacterial agents of disease…..’ This general issue was discussed in the Searching for Molecular Solutions associated ftp site, in the Cited Notes for Chapter 3, Section No. 4 (DNA-binding protein design).
Note: If problems are encountered in directly accessing the above ftp site, go here, and then click on molecular-solutions.zip (8.11 Mb).
This post and the succeeding one are the culmination of a recent theme dealing with ‘Natural Molecular Space’ . This series has looked at the biology of natural products, and also their use by both animals and humans from the earliest of times. Here the same activity in the modern world is considered.
Natural ‘Chrestomolecules’ Now vs. Then
An earlier post discussed the significance of ‘chrestomolecules’, or useful molecules of economic significance (in its broadest sense). This survey did not discriminate on the basis of any molecular properties, and a range of different categories of such molecules was listed. Yet ‘natural molecular space’ as considered in several previous posts (19 July, 26 July, 9 Aug, 16 Aug) has been preoccupied with small molecules. How does the “old” (traditional) vs. “new” (modern) use of all molecules from the biosphere stack up if viewed through a size-based lens?
Notes for diagram: This compares traditional and modern use of all bioproducts. The traditional group is encompassed as a subset of the wider modern group, with Nutrients as a special category. Sources based exclusively on small molecules are shown in blue; those based on either large or small molecules are shown in red, and those based on large biomacromolecules (mostly proteins and nucleic acids) shown in orange. ‘Repetitive biopolymers’ refer to molecules of biological origin whose structures are usually highly repetitive polymers based on a limited number of subunits, such as certain polysaccharides. ‘Therapeutics’ is placed in both Traditional and Modern groups, owing to the use of many small natural biomolecules within each, but therapeutic antibodies only within modern times.
The above diagram uses the categories found in the chrestomolecule Table of a previous post, and sorts them into two broad levels based on their histories of usage. The main message is the preponderance of small molecules in traditional applications. With the exception of certain biopolymers of relatively regular (and often linear) structures, the traditional group does not feature complex proteins and other macromolecules which are well-represented in the modern set. Why should this be the case? Clearly, knowledge and technology (only available from relatively recent times) make a big difference, in allowing the exploitation of the properties of large and complex molecules. Also many small molecules have an inherent major advantage over potential protein therapeutics in terms of their oral delivery potential. Traditional societies could not provide intravenous delivery by hypodermic syringes, and protein or peptide-based substances usually fare poorly in making transit through the acidic digestive barrier of the stomach.
So, this size-wise breakdown will be used as a divide for the coverage the modern use of the biosphere: This post will feature the modern harvesting of natural small molecules, and the next will concentrate on the modern use of natural large molecules, and indeed entire useful molecular systems.
What other general differences might then exist between the (very) old and the (relatively) new, in this context? As a broad principle, one could note that modern science, technology, communications and mobility provide the ability to initiate systematic screens of a wide variety, with increasing refinements as to exactly how they are performed. One consequence of this is the ongoing exploitation of marine environments for natural products, which were almost untouched during ancient times. This theme is looked at in a little more detail below. Another area unique to modern times might be summed up by the phrase ‘getting more from what’s on offer’, or using natural drug precedents to the best advantage through technological developments.
Harvesting of Small-Molecule Natural Molecular Biodiversity in Modern Times
At this point we have considered natural molecular space from a number of viewpoints, including its functions, evolution, classification, and empirical exploitation by traditional tribal medical lore. This leads us to a central issue directly relevant to the theme of this post: How important do natural products as a whole remain for human needs, to what extent have they been replaced by other technologies, and what are future trends in this area of biotechnology? Or using a previously-introduced terminology, what fraction of chrestomolecules now and in the foreseeable future will derive from natural biomolecules?
Beyond two decades ago, a majority of pharmaceutical drugs derived from natural product sources, but by the 1990s the fraction of drugs directly or indirectly originating as natural biomolecules stood at about 50%, owing to advances in synthetic chemistry and combinatorial screening. Nevertheless, the fraction of naturally-derived pharmaceuticals varies considerably if specific therapeutic applications are considered. Over the time-frame 1981-2006, over 70% of cancer drugs have been cited as non-synthetic in origin, and naturally-derived drug percentages increase in all therapeutic categories if one includes semisynthetic analogs based on the original core structure of the biomolecule. In this area, the use of natural product molecular scaffolds for the future design of antibiotics has been promoted. Natural products remain highly regarded for their diversity and as source of novel structural motifs. Generalizations of the properties of natural product molecules have shown significant average differences to synthetic molecular libraries, including steric complexities, atomic contents, and ring structures. As considered in a previous post, the evolutionary origins of natural products may provide a positive bias in favor of their utility, at least as a source of novel molecular scaffolds. Even among plant sources alone there is still a huge range of material remaining to be investigated by systematic ‘bioprospecting’.
In modern times, conscious attempts have been made to harness traditional ethnic knowledge of therapeutic natural products, with the aim of accelerating drug discovery. As noted previously, these kinds of studies have been termed ‘ethnobotany’ (given the preponderance of plant products involved) or more generally ‘ethnopharmacology’, and the advent of a number of important drugs have been attributed to the transfer of ethnopharmacologic knowledge. (The example of quinine, and the complexities associated with Europeans’ awareness of it, was considered in the previous post). The continuing value of ethnopharmacology has been vigorously promoted by certain researchers. Throwing cold water on this enthusiasm to some extent, over a twenty year period the US National Cancer Institute (in the course of systematically screening very large numbers of plant extracts) did not find useful anti-cancer drugs specifically from ethnobotanical information, with the possible exception of the anti-cancer drug taxol.
Commercial interest in ethnopharmacology in recent times has led to the formation of companies dedicated to mining such potentially valuable knowledge as the basis for a drug discovery platform. One much-publicized example is the now defunct Shaman Pharmaceuticals, but numerous companies have had at least a passing interesting in drug acquisition by such means. A highly contentious issue has been the rights of indigenous peoples to compensation if their information led to a successful profit-making venture, and accusations of ‘biopiracy’ have been made towards many Western drug discovery activities in environments used by native people. These kinds of political, legal and ethical issues have clouded or retarded a number of relatively recent bioprospecting ventures. Despite the importance of these considerations, a more fundamental problem is the continuing destruction of rainforests and other natural habitats, which threaten to result in irreversible losses in biodiversity and rich sources of novel biomolecules. Associated with this, and the forces of cultural homogenization, loss of tribal languages and lore are also lamentable outcomes not only in their own right but as a potential source of ethnopharmacological information. This is an absolute loss if the ethnic group is pre-literate, but if old written records exist they may possibly be tapped for such irreplaceable knowledge.
A Natural Frontier in the Sea
In contrast to terrestrial environments, the seas and oceans have not yielded a large number of notable traditional medicines, or an associated rich ethnopharmacologic folklore. This is undoubtedly due to the relative inaccessibility of most marine environments without comparatively recent technological back-up, and marine bioproducts are thus greatly under-represented in traditional medicinal tool-kits. Even peoples with close access to the sea, such as the Samoans, appear to have derived most of their traditional ethnopharmacological lore from land plants. The environmental ‘diversity factor’ D noted in the previous post for traditional drug discovery is in turn reduced in practical terms by the inability to recognize and ‘fish’ the oceans for useful molecules. Thus, the marine environment has been poorly exploited as a source of drug discovery until recent times, despite it bearing a plethora of a potentially useful and highly diverse organisms with equally diverse biosynthetic capabilities. Or possibly even more; it has been claimed that at this juncture in history, searching marine natural molecular space is much more likely to yield novel biodiversity than land ecosystems.
Sponges alone have proven to be rich in a variety of bioproducts with promising applications. Useful metabolites, possibly with antimicrobial properties, may be obtained from seaweeds or marine cyanobacteria. Conotoxins (from cone shell molluscs) from approximately 500-700 Conus species are a highly diverse family of peptides with neurotoxic activities. These find many important applications in neurological research and possibly in a number of therapeutic contexts, at least as prototype molecules pointing towards pathways for future drug development. Sea hares, marine molluscs which have found productive application in research on memory mechanisms, are yet another source of useful products, including antibacterial proteins. Tunicates (sessile marine invertebrates) have yielded chemically diverse cytostatic and cytotoxic drugs with potential applicability in clinical oncology. Given the continuing unmet need for effective anti-cancer treatments, and the very large international market which successful cancer therapies can fill, it is not surprising that commercial interest has been stimulated towards marine natural products with potential for this kind of activity. Numerous clinical trials for the anti-cancer efficacy of certain marine bioproducts have been conducted and are continuing.
In a number of cases of apparent production of useful compounds by marine invertebrates, the true source may be commensal micro-organisms carried by the invertebrate organism. Combining this observation with the relatively poor knowledge base concerning marine microbes, and their ancient and robust variety, assessment of marine microbial populations accordingly deserves high priority for the analysis of oceanic biodiversity. A high-profile expedition launched with this end in mind has been the ‘Global Ocean Sampling’ voyage, under the aegis of J. Craig Venter, his eponymous Institute, and other participants.
More Bang for Your Natural Product Buck
At the present time conventional means for identifying and optimizing natural products has been supplemented by a number of different approaches, with some major examples shown in the Table below. Let’s now examine these in a little more detail.
Recent and ongoing advances in identifying, screening, processing, and developing natural products.
The first six categories of this ‘improvements’ Table deal with better ways for finding natural products of interest in the first place; categories 7 and 8 are more concerned with modifications and betterment of candidate molecules in hand.
Natural products are initially encountered as complex mixtures, and thus efforts have been made towards streamlining sample preparation and purification as much as practicable (No. 1 of the above Table) in advance of screening. Included within this category are important advances in the determination of the molecular structures of natural products from extremely small sample sizes. The capability of evaluating candidate samples for specific properties in very high volumes and with great rapidity (high-throughput screening) is also an important issue in modern natural product evaluation.
With the rise in bioinformatics and computer modeling, ‘virtual screening’ (No. 2 of the above Table; equivalent to computer-aided evaluation of possible candidate drugs by modeling their interactions with target receptors) has become an important adjunct in bioproduct testing, as well as general drug identification. Bioinformatics is also applied in computational searching for new members of specific gene families (such as novel biosynthetic genes), which may act as drug targets, and an important pre-requisite for this is the availability of complete genome sequences for an increasing number of key organisms which produce secondary metabolites.
Empirical screening of any bioproducts is fundamentally dependent on a specific assay and its read-out, whether it is high-throughput or not. If one is seeking a compound which can usefully modify a particular cellular system, a good understanding of the underlying biology of the system is likely to identify the specific molecule(s) which should be targeted. This in turn is a clear advantage for screening itself. Refined understanding of fundamental cellular processes involved with carcinogenesis, for example, will in general lead to assay improvement (No. 3 of the ‘improvements’ Table above). Ultimately, with a single-molecule target and structural information, a rational design strategy may become possible. Prior to such a point, a natural product (or any other) molecular library will be best put to use if it is well-focused on the appropriate target. While less efficient, a more complex assay system (such as whole cells) may on the other hand provide additional information about other side-effects of the tested compounds. The utility of therapeutic bioproducts is critically dependent on their abilities to approach the ideal of ‘magic bullets’ in complex biosystems, without deleterious side-effects.
The next item (No. 4) in the above Table refers to metagenomics, which has been briefly discussed in a previous post. In this context, the relevance of metagenomics centers on the large fraction of environmental bacterial species which are non-cultivatable in the laboratory, but which could potentially yield useful drugs. Amplification and assembly of environmental DNA samples into discrete genomes (in essence, the ambit of metagenomics) has the potential to accelerate the analysis and manipulation of important and novel metabolic pathways, through which new small molecules are synthesized. Accordingly, rapid high-throughput genomic sequencing technologies (No. 5) available only in past few years feed into this process. Further manipulation of whole pathways, ultimately with entire synthetic genomes (the domain of synthetic biology, item No. 6 of the ‘improvements’ Table; and discussed generally in a previous post) in turn will provide great control over both the production of new active secondary metabolites, and their specific chemical modifications as desired. These ambitions require an integrated ‘systems-level’ understanding of the pathways involved in the production of all small molecules by an organism of interest, or its metabolome. High-level metabolomic input thus has great potential for the engineering of organisms for either increased yields of specific secondary metabolites, or the production of novel ones.
Since early times in antibiotic research, natural products have contributed core molecular designs (‘scaffolds’) which have been modified artificially, either by complete or partial synthesis (No. 7 of the above Table). This effort towards bioproduct improvement is still a productive venture. The final (eighth) innovation of the ‘improvements’ Table above concerns attempts to improve yields of metabolites by chemical ‘elicitors’, and also efforts towards boosting metabolite chemical diversity by modifying culture conditions.
These modern technological developments, combined with the well-noted evolutionary advantages of ‘consulting’ the natural compendium of biological small molecules, suggests that there is still much practical value to be gained from them. The question of the ongoing future of the exploration of natural molecular space will be picked up again in the next post, particularly insofar as it may be eventually superseded (at least in part) by wholly artificial alternatives.
And finally, a general comment on the importance of technology in the modern science of natural bioproduct discovery and development, in a biopolyverse-like manner:
Bioprospectors don’t need to carry picks
Since they have a range of techniques in the mix
Using various means
And comprehensive screens
They can rely on a complete bag of tricks.
References & Details
(In order of citation, giving some key references where appropriate, but far from an exhaustive coverage of the literature).
‘…..small molecules have an inherent major advantage over potential protein therapeutics in terms of their oral delivery potential….’ It may be noted that this general issue raises the whole field of drug delivery, the science of devising the means for ensuring that useful drugs reach their intended in vivo targets in an efficient manner while retaining their functional properties. This is a huge modern field, of the most fundamental significance to pharmaceutical companies.
‘…..the fraction of drugs ….. originating as natural biomolecules stood at about 50%…’ See Li & Vederas 2009.
‘….over 70% of cancer drugs have been cited as non-synthetic in origin….’ See Newman & Cragg 2007.
‘…..the properties of natural product molecules have shown significant average differences to synthetic molecular libraries….’ See Koehn & Carter 2005.
‘…..Even among plant sources alone there is still a huge range of material…..’ See Phillipson 2003.
‘..…the advent of a number of important drugs have been attributed to the transfer of ethnopharmacologic knowledge….’ See Cox 1990.
‘……the US National Cancer Institute ….. has not found useful anti-cancer drugs specifically from ethnobotanical information…..’ See Cragg et al. 1994; ‘….with the possible exception of the anti-cancer drug taxol…’ See Cragg 1998.
‘…..accusations of ‘biopiracy’……..’ See Shiva, S. Biopiracy: the plunder of nature and knowledge (South End Press, Cambridge MA, 1997); ‘……..have clouded or retarded a number of relatively recent bioprospecting ventures……’ See Rosenthal 2002.
‘…..peoples with close access to the sea….appear to have derived most of their traditional ethnopharmacological lore from land plants…..’ See Cox 1993.
‘……marine natural molecular space is much more likely to yield novel biodiversity than land ecosystems….’ See O’Hanlon 2006.
‘ Sponges alone have proven to be rich in a variety of bioproducts with promising applications….’ Sample references for modern harvesting of marine bioproducts: From sponges, see Sipkema et al. 2005; seaweeds, see Kubanek et al. 2003; marine cyanobacteria, see Burns et al. 2005; conotoxins, see Alonso et al. 2003; sea hares, see Barsby 2006; tunicates, see O’Hanlon 2006, Simmons et al. 2005.
‘…..the ‘Global Ocean Sampling’ voyage….’ See Rusch et al. 2007.
‘…..advances in the determination of the molecular structures of natural products from extremely small sample sizes….’ See Murata et al. 2006.
‘……(high-throughput screening) is also an important issue…..’ See Koehn & Carter 2005.
‘…….‘virtual screening’ …..has become an important adjunct in bioproduct testing……’ See Rollinger et al. 2008.
‘……an integrated ‘systems-level’ understanding of the pathways involved in the ….. metabolome….’ See Weckwerth 2010. A good example of the economic significance and challenges faced with metabolic pathway manipulation can be found with efforts to engineer the synthesis of the anti-malarial drug artemisinin in microbes for large-scale production. Of Chinese origin, this compound has been effective against the deadly Plasmodium falciparum malaria species, but supplies of its natural plant source (Artemisia annua) are often limiting. To engineer ‘heterologous expression’ of the drug in microbial cells, an entire pathway of enzymes must be provided within the foreign host cells. To date, successes with producing artemisinin precursors in yeast and E. coli cells have been reported. See Arsenault et al. 2008 for an overview, and Tsuruta et al. 2009 for details on an E. coli expression system.
‘……to improve yields of metabolites by chemical ‘elicitors’…’ See Poulev et al. 2003.
‘…..efforts towards boosting metabolite chemical diversity by modifying culture conditions….’ See Bode et al. 2002.
‘…..suggests that there is still much practical value to be gained from them [small natural bioproducts]’ See Li & Vederas 2009.
Next Post: Two Weeks from now.