Predispositions to toxic risk; Moderators of toxic risk; C 4, 9; Carcinogenesis : Are the toxicity models correct?; C 8, 9, 25

Discussions & Projects

Hopefully the Prometheus Site Administrator and I have ironed out most of the problems we ran into last week with the posting of lecture materials. I would suggest for your own future project postings that when you construct the zip files, you do not make them self-extracting. I do not know if that is a default for WinZip, but whenever I have looked at those projects that have been zipped using that program, the file fails to open within the Prometheus site. Instead it downloads and opens in the host computer. Workable, but not optimal.

There is a lot of good commentary coming through on the projects in the Discussion area and I'm seeing some threads being picked up and worked through for the Discussion questions as well -- that's what's supposed to be going on there! Please keep adding to what has been posted. If you have not already commented on the projects, make sure you do so (it's part of your grade). Also, make certain you review those comments and try to adjust your projects accordingly. I will be posting my own comments on the projects and on a number of the Discussion questions in next week's posting.

Predispositions to Toxic Risk

While I included the next statements earlier in the term, it may be well to reiterate them in the context of this session as we begin to look at the use of dose assessments and biological test or epidemiological data to assess risks of toxicant exposure.

The business of much of toxicology is the evaluation and establishment of the type and degree of risks posed by exposures to particular toxicants or toxins. Risk assessment is a set of procedures intended to look at the cost/benefit ratio of exposure versus nonexposure, or exposure versus lesser exposure. It involves a detailed examination and evaluation of alternatives to exposures and the various potential costs involved in exposure versus those alternatives. The alternatives are not necessarily simple substitutions, they may be entirely different solutions to a given technical or social problem, they are often "apples and oranges" such as economic benefit versus medical or ecological risk. Such evaluations are often decided based on a set of contrasting counter arguments with the most logical or powerful debater in a given dispute prevailing over those with less strong arguments or information. As such, these debates often have no "right" or "wrong" answer.

There is no doubt that risks will vary due to variations in the levels of exposure across a population. But even given similar dosages, some individuals within a population will be at higher risk to demonstrate the biological consequences of exposure. Most of this is traceable to differences in physiology and metabolism which, in turn, are dependent on expression of underlying genetic potential. Clearly, for example, there are gender differences in metabolic capacity or hormonal "climate" that are expressed in varied susceptibility to the negative impacts of certain chemicals. Breast cancer is preponderantly a "female" problem while prostate cancer is a "male" problem; clearance rates for many chemicals differ between males and females as do occurrences of non-reproductive cancers. Ethnic differences in sensitivity to ethanol consumption or to ultraviolet light exposure also reflect genetic variation across populations. In addition, however, there are individuals who have inherited weaknesses in immune or DNA surveillance or repair mechanisms that make them particularly prone to the development of negative responses to certain toxicants.

A classic example is familial retinoblastoma. Individuals who have inherited one mutated allele of the Rb gene (the first major cell-cycle regulatory protein) have only one wild-type allele. They are particularly prone to development of retinal, lymphoid, and several other cancer types in childhood or adolescence because only a single mutational event is required in their cells to compromise a major control on normal cell division. Natural light exposure of the retina provides a mutational challenge that can readily produce a mutated, and now unregulated cell. Those chemicals capable of mimicking the effects of radiation will similarly challenge familial RB subjects.

The increasing incidence of asthma in urban youth unquestionably reflects differences in exposures to air-borne chemicals associated with urban life. However, it almost as assuredly also reflects some subtle shifts in immune competence within the populace as a result of improved medical care and a lack of exposure to natural allergens early in life. More mildly immunocompromised individuals persist to reproductive age in the population than would have with less access to modern medicine. And even completely "wild-type" immune systems are less challenged when living in regulated air environments than they would have been in earlier times thereby reducing the somatic fitness of the average immune system. Under such conditions, chemical toxicants can have impacts that would not have been possible in earlier times; they create more susceptible populations.

Life-style factors can also contribute to risk predispositions. Residence in areas where natural concentrations of uranium occur will predispose to exposure to excessive radiation. Smoking will sensitize air passages to other air-borne toxicant including the radioactive decay products of radon. High stress occupations suppress immune responses and elevate susceptibility to toxicants as well as pathogens.

All such situations predispose these individuals to toxic risks. Their individual dose-response curves for a particular toxicant will be shifted left (toward lower doses) than those of the rest of the population. They form the low-dose tail of population dose-response curves.

Moderators of Toxic Risk

On the other hand, high fiber diets mitigate consumption of food-associated toxicants by altering, among other things, enterohepatic circulation, the ability of lipophilic chemicals to diffuse into the mesenteric blood supply, and the overall redox state of the organism. European parentage tends to increase the capacity to detoxify ethanol. Experiencing full-term pregnancies is protective against many female reproductive tract and breast cancers and, apparently, the environmental hormones that may tend to promote them. And living in the rural Midwest decreases exposures to many industrial solvents.

Many such genetic, epigenetic, or life-style variations will tend to be protective against the risks of intoxication. As opposed to the predisposed populations, the protected groups will experience lower biologically available toxicant dosages for any given level of exposure and/or they will have actually lower exposures associated with the form of the variant. These will be the individuals with a right-shifted individual dose response curve. They will be at the high end of the population dose-response.

But note I titled this section Moderators of Toxic Risk. Co-exposures, or dietary, medical or other conditions can also increase risks for more limited times than the lifetimes noted for predisposition to toxic risk. Under those circumstances risk will increase coincident with the modulating influence. Combining ethanol exposure with barbiturates prolongs barbiturate effects and increases risks of intoxication.

So assignments of risk always describe distributions rather than specific numbers. This must be recalled in reading much of the literature on risk assessment. It is an inexact science and the precision of many of the computations cannot capture true levels of risk unless reported along with some a discussion of how the ranges of risk values vary across populations at risk and what factors are particularly important in altering the levels of risk assigned.

Carcinogenesis : Are the toxicity models correct?

The Traditional Carcinogenesis Model:

The definitions given by C&D tend to rule out endogenous hormones as being classed as carcinogens unless they are exogenously administered. But what about endogenous overproduction of trophic or growth factors? Or incidental exposures through food and the environment?

The toxicology text provides a disappointingly naive discussion of endocrine systems given the similarities of toxicology, pharmacology, and endocrinology with respect to methods and many of the central concepts concerning mechanisms of action, chemical kinetics, clearance, and general physiological function.

The current carcinogenesis model looks at three stages in the "natural history of neoplasia:"

1. Initiation (primary molecular lesion formation, adducts, mutation; reversible)

2. Promotion (enhanced ability to escape normal controls for removing adducts or mutations and to allow abnormal cell proliferation; may still be reversible)

3. Progression (overt cell transformation, nuclear instability coupled with loss of mitotic controls and metastatic behavior; irreversible)

A chemical may play a role in the actual induction of mutation or the initial stages of neoplastic transformation in the first cell involved. It may play a role in preserving that cell and making certain it can continue to divide. Or it may play a role in allowing the initial cell mass to invade other tissue and/or to metastasize to other sites. If the model is correct there should be examples of chemicals among the thousands examined that can be identified with each of these steps. Yet there are few, if any, that can unequivocally be assigned to initiation and progression. This would suggest there may be a problem with the specification or design of the current model. Or it may simply be a reflection of our current ignorance about the mechanisms by which many of these chemicals exert their toxic effects.

Why screen breast cancers for estrogen sensitivity if hormones are not directly involved in initiation of neoplastic growth as suggested by Casarett and Doull?

Breast tumors are screened for the presence of estrogen receptors because estrogen acts through such receptors to promote expression of those genes that lead to cellular replication via mitosis. Estrogen is pro-mitotic in many of the tissues in which it normally acts. Thus, knowing if a tumor is sensitive to estrogen can provide a means to suppress tumor growth. Aromatase inhibitors can be given to suppress ovarian (if a pre-menopausal woman is involved) or peripheral production of estrogens. This will often slow the tumor growth or its production and response to paracrine growth factors like EGF, or TGF . In turn, this provides more opportunity to apply radiation, surgery, and/or directed chemotherapy agents to ablate the tumor. Obviously, if the tumor does not have estrogen receptors, it will not respond to such treatments.

Likewise, testosterone or dihydrotestosterone are pro-mitotic in male secondary structures like the prostate. Here anti-androgens can be applied to good effect so screening could be used for some tumors of the prostate. (Normally slow progression of these growths and the lack of ready anatomic access to the prostate tends to make this a less frequently used approach.)

Casarett and Doull point to the actions of growth hormone and other growth factors and suggest these may be considered chemical carcinogens. This is contrary to the definition of toxicants and really only fits the notion of toxins except that both refer to substances administered exogenously. Certainly hormones are not exogenous. Their actions at the high levels characteristic of endocrinopathies may eventuate in neoplastic conditions. Whether they are truly the initiators of these neoplasias can probably be questioned (environmental factors, exogenous radiation, etc.). Model systems such as knockout or overexpression models do, however, suggest that hormones can, if not initiating cancers, at least act as sensitizing or predisposing agents. Such is probably the case for the elevation of dimethylbenz[a]anthracene (DMBA) tumor formation seen in rats expressing high levels of prolactin. Both estrogens and prolactin are mitogens in rat breast tissue. Their actions will increase the number of cells undergoing division in any given time. If DMBA is applied simultaneously, or subsequent to this action, the intercalating agent DMBA will have additional opportunities to disrupt the crucial machinery involved in DNA synthesis and repair associated with mitosis. That does not make estrogen or prolactin co-carcinogens, but it does mean that they will predispose responsive tissues to agents that interact with DNA. The same situation will also apply to dimethylnitrosourea application to prostate tissue simultaneous with or subsequent to promitotic androgens such as testosterone. In fact, it should apply in any situation where a hormone alone stimulates a tissue to increase mitotic activity.

Note the dates of the references in Table 8-2 concerning the development of tumors subsequent to disruption of normal hormonal feedback cycles. Certainly there has been work done in these areas over the past decade that may be pertinent to these discussions. Hormones and their control circuits are natural, homeostatic mechanisms. They are only carcinogenic if the stimulation of cell division processes, especially mitosis, becomes disrupted due to high rates of division coupled with failures of checkpoints or apoptotic controls and immune surveillance that normally remove aberrant cells. We think of carcinogens as inducers of transformation/ mistakes/ mutations. Hormones don't do that, thus, they are probably best classed as promoters, or possibly supporters of progression.

Alternative Views of Carcinogenesis:

The text authors also seem to hold a somewhat dated understanding of how hormones and genes play roles in neoplastic and cancer development and growth. I would definitely refer you to the publications of Drs. Robert Weinberg of the Whitehead Institute at MIT and Polly Matzinger of NIH with respect to critical factors involved in the processes of cellular transformation and immune surveillance associated with cellular transformation, respectively. Several pertinent files or articles are linked to these lecture notes.

counteretalPNAS98.pdf

dangerturn.shtml

jacks&weinbergscience98.html

Lees&WeinbergPNAS99.pdf

lundber_weinberg98.html

orrweaver&weinberg98.pdf

polly.html

weinbergNature.pdf

Weinberg in a lecture to the Endocrine Society in Denver in 2001, mentioned at least four cellular features that had to be altered before a cell could undergo transformation from a normal to a cancerous cell. First, mutations or alterations had to occur in one or both the the cellular mitotic checkpoint proteins, pRb, and p53. Second, a change in expression of proteins involved in the apoptotic pathways had to occur so that this process was either not initiated or was actively blocked. Third, telomerase had to be expressed so that the cell could overcome the limitation on division normally encountered due to shortening of the telomeres in each round of cellular division. Normal differentiated cells express little or no telomerase. Transformed cells express significant levels. Finally, to actually form a tumor, the transformed cell has to avoid immune surveillance.

The latter point is probably best addressed by Matzinger's work which posits that the immune system is not active as normally thought, but rather, reacts only to the products of cell death. Either released cytokines or chemicals that ultimately cause production of immunoattractants like leukotrienes or interleukins (or possibly released cytochrome c?).

Note that the complex of steps required for expression of full-blown carcinogenesis means that the frequency of truncated events that fail to reach this level is probably considerable even under normal circumstances. The elevations in this basal rate is what can be associated with carcinogenic toxicants.

Review the description of proto-oncogenes, oncogenes, and oncogene products that occurs in an endocrinology or cell biology text. Note that proto-oncogenes are normal genes, they are not strictly associated with transformation. Oncogenes are normals genes that are inappropriately expressed with respect to developmental timing or level. They may be, but do not have to be, altered structurally. In fact, many are structurally normal, but are altered in genetic position or in their regulatory domains so that they are inappropriately expressed. Those that have been described often include elements of transmembrane signalling pathways associated with cellular growth and division. Note the proto-oncogenes or the oncogenes themselves may be quite neutral in cellular transformation; what is important is the inappropriate expression and subsequent actions of the oncogene products. If chemicals cause alterations in DNA synthesis or repair that result in mutations to proto-oncogenes or their regulatory regions, the result is the oncogenic expression of the altered proto-oncogene (now referred to as an oncogene). The only other route to this condition is a toxicant alteration of the expression of a particular proto-oncogene (or a set of such genes) so that it acts as an oncogene. This might be via a targeted mutation or a specific alteration in the biochemical pathways leading to expression (or suppression) of that particular gene product.

Note that the above view of the integration of proto-oncogenes and oncogenes into cellular physiology seems to differ from the picture of the "foreign invader" provided in the text. It views cellular transformation as a natural process exacerbated by the presence of toxicants, not as a novel process imposed on the system by the presentation of a toxicant.

Radiation Hazards

Some of the basics on radiation were covered during Session 4. This coverage will only touch on a few additional items.

The question arose, given the cautions regarding food additives or composition across species as complicating issues in toxicity evaluations, as to whether, as claimed in some lay literature, microwaved food was a hazard during pregnancy and gestation.

Evidence is slim that the limited microwave exposure caused by waiting outside a microwave would have a direct effect on mother or fetus. Moreover, save for heat, the microwave -- unless the food exposue is so prolonged as to cause charring -- will not leave a residue in the food that might make such food hazardous. Rather, it is more likely that the suggested avoidance of microwave foods during pregnancy is more based on their typically higher content of salt and preservatives as nutritionally less adequate than any exposure of the food or the cook to microwaves.

But this raises the more general question of whether irradiated food, e.g., gamma irradiated meat or milk, is safe for consumption?

It should be safe because the irradiation is in the past relative to presentation to the consumer. Radiation does not leave residues or residual radiation in the food. Moreover, the act of irradiation kills the degradative organisms in the food that could cause formation of noxious or toxic products; it should make the food safer rather than less safe. Finally, empirical evidence, lack of negative or untoward effects in areas where irradiated food is used, suggests such food is safe.

Is there anything that might make it unsafe? Only if the irradiation is sufficiently strong or prolonged to cause chemical changes that generate toxic byproducts in the food. More likely such food would become physically "burned" or otherwise undesirable in addition to becoming "less safe." Note there is little if any empirical evidence indicating that such byproducts are encountered during the routine processing of foods for consumer consumption.

Coverage in C&D on radiation as a toxin fails to make several important points. First, radiation can have disruptive effects short of the induction or carcinogenesis or death. For example, electromagnetic radiation including x- and -rays or proximally applied particulate radiation are effective means of inducing sterility in both males and females. They are capable of producing burn-type lesions at high dose rates – exactly the basis for their use in chemotherapy or localized tissue ablation. Second, radiation resembles many chemicals in its impacts on cellular physiology. Many effects are due to the same kinds of disruptions of cell cycle machinery and recruitment of repair mechanisms that occur with chemicals that interact directly with DNA. Because of this overlap, the discussions of risk assessment covering radiation are good models for similar assessments concerning chemicals that are radiomimetic. Moreover, the coverage of background/environmentally encountered radiation provides a particularly good example (as radiation is relatively easily quantified) of the complications of assessing toxic risks in the presence of naturally existing, ecological, contamination.

The coverage does present at least one case of meta-analysis. In a meta-analyis, qualitatively similar studies, often of populations, are analytically combined so that the statistical power of the combined study can be used to detect effects that were impossible to see in each of the individual small studies. At least that's the idea. Unfortunately, the slight differences among studies or among the populations studied often inflate the errors associated with parameter estimates so that some of the gain in statistical power is lost during the re-analysis. In some studies that loss compromises the overall impact of the meta-analysis, in others there is a sufficient gain to demonstrate what could not been proven in the smaller studies. The approach is an efficient means of exploring questions for which ample collective data exists but which would be prohibitively expensive to undertake de novo.