|Some natural and synthetic chemicals are genotoxic because they react directly with DNA or its associated proteins to form strongly-bound (covalent) adducts. The most frequently-studied endpoint of genotoxicity is cancer. In the document proposed here, the genotoxins discussed are electrophilic compounds, which are chemicals that react with electron-rich (nucleophilic) sites on the biological macromolecules. Sometimes a chemical to which one is exposed may be intrinsically unreactive, but is metabolised to toxic electrophilic products in the liver and other organs. Some normal mammalian metabolites are genotoxic, at least in theory.
Much research is aimed at identifying potentially genotoxic compounds to which humans may be exposed. One of the objectives is to estimate the proportion of cases of cancer that can be attributed to such exposures. While the proportion of cases due to ionising radiation can be estimated, there is still much uncertainty about all other causes of cancer, which include infections and spontaneous metabolic errors. Over the years, I have come across a few possible examples of potential chemical hazards that, in my opinion, do not seem to have been evaluated adequately.
Chemical methods have been used to estimate the genotoxic potency of chemicals suspected of being hazardous; biological test methods are indispensable but they are expensive and not always sensitive enough. Some chemical methods allow one to explore the mechanisms of the reactions between electrophiles and DNA. I noted that most applications of the most popular of these methods (NBP), which is also used for analytical purposes, have serious deficiencies with respect to the design of experimental protocols, and consequently to the interpretation of results. The difficulties may be attributed to scientific misunderstandings in an exceptionally multidisciplinary field. Following discussions with former colleagues I attempt to give some background information that is not often provided in a suitable form to scientists working, for example, on genotoxic drug impurities.
The document is neither comprehensive nor authoritative, and it has been kept as informal as possible; suggestions for modifications are welcome.
The abstract is given below.
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|Medicinal products sometimes have adverse effects that concern a small but significant proportion of patients. Since a person can fall ill at any time, special statistical analyses of large numbers of adverse event reports are needed to establish whether a particular type of symptom could have been caused by a particular treatment. Worldwide pharmacovigilance programs exist for this purpose. Detecting quantitatively minor genotoxic causes of common types of cancer would be particularly difficult, because the background incidence is high and variable, and symptoms appear a long time after exposure. Anticipatory testing may not be sufficiently sensitive to detect genotoxicity risks that are considered significant (officially, one case per 100~000 patients on the basis of life-long drug treatment). While carcinogenic risks due to ionising radiation have now been established for exposures down to those encountered with diagnostic x-rays, it is rarely possible to make similar calculations for exposure to chemical carcinogens, even when the existence of a hazard is known.
Many natural and artificial carcinogens have been identified, and they are considered more or less seriously, depending on public and personal attitudes to them. The subject of this document is the possibility that further significant exposures to genotoxic (and therefore likely carcinogenic) compounds of synthetic origin may have been either unsuspected or ignored.
Three possible examples of potential pharmaceutical genotoxic hazards are presented, and the third of these may have ramifications for non-pharmaceutical applications of the products concerned. In each of these cases, it could be envisaged that chemical compounds may react with DNA in a manner that is known to have genotoxic consequences.
1. Unexpected chemical reactivity in a series of drug substances having the ortho-alkoxybenzamide structure is alerting for genotoxicity.
2. Various nitro-heterocyclic antibiotics have known alerts for genotoxicity; they are banned for administration to food animals but not to humans. However, in view of the proliferation of microorganisms resistant to other antibiotics, these products could possibly provide leads for new products for use as a last resort.
3. The semi-synthetic carbohydrate ethers, widely used as drug excipients and for many other purposes, may present a genotoxic hazard that does not seem to have been discussed.
It is likely that cases such as these could be brought to light only by some form of chemical serendipity. Unfortunately, there does not seem to exist any official body to which scientists may report such concerns in a responsible manner. A probable reason for lack of attention to this subject is its interdisciplinary nature; no-one feels confident enough to discuss and evaluate every aspect. No-one is able to estimate the risk associated with a potential hazard that has not yet been investigated, nor to decide whether a question merits investigation.
Some background information is given in an appendix:
- The chemistry of relevant types of chemical reactions of xenobiotics with DNA is presented, partly to demonstrate serious apparent deficiencies in some published research and review papers. Numerous studies have attempted to correlate electrophilic reaction kinetics and mechanism with DNA adduct formation and consequent mutagenesis. The reagent 4-(4-nitrobenzyl)pyridine (NBP) is commonly used as a surrogate for DNA, but much reported experimental data is questionable because of chemical artefacts that have been described but are generally neglected.
- Some electrophilic mutagens that react with DNA have linear dose-response relations. Others, including many to which humans can be exposed, have different reaction mechanisms; they show non-linear responses indicating relatively small effects at low doses. Consequently, they are considered to present risks that are lower than would be estimated for a linear relation. The difference has been ascribed to (demonstrated) saturable DNA repair activity, which is more or less effective depending on the sites of DNA-adduct formation; site-specificity is a function of the reaction mechanism. Surprisingly, despite the importance of the subject for the safety of medicines and other chemical products, the literature on DNA repair does not seem to have been evaluated in the context of known toxicokinetic factors. In particular, depending on the reaction mechanism, an electrophile may or may not be scavenged in a saturable manner by endogenous nucleophiles.
Table of contents
1.1 Genotoxicity and cancer
1.2 What cancer risks are considered acceptable?
1.3 Relating exposure to a genotoxic chemical to the risk of cancer
1.4 Regulatory and other influences
1.4.1 Note on the literature on electrophilic reactivity and
2 The ortho-alkoxybenzamides
2.1 Reaction mechanism; natural N-methylation of DNA, and DNA repair
3 Nitrofurans and other nitro-heterocyclic drugs
3.0.1 Why isn't lower-intestinal cancer even more prevalant?
3.0.2 Could a genotoxic mode of action be a starting point
for developing future antibiotics?
4 Carbohydrate ethers
4.1 Manufacture, impurities and uses
4.2 Chemical reactivity of carbohydrate ethers
4.2.1 Intrinsic reactivity
4.2.2 Reactions leading to formation of reactive alkylating
agents in drug substances
4.3 Possible formation of reactive alkylating agents in formulated
4.3.1 Notes on potential pharmaceutical hazards
4.4 Potential hazards of carbohydrate ethers in non pharmaceutical
4.4.1 Tobacco, and other applications involving pyrolysis
5 Summary and conclusion
A Alkylation of DNA, and genotoxic endpoints
A.1 DNA adduct formation by electrophilic reagents
A.1.1 SN1 and other non-selective reactions
A.1.2 SN2 reactions
A.1.3 Mixed SN1/SN2 mechanisms
A.1.4 Notes on DNA reaction sites
A.2 Predicting and evaluating reactivity and selectivity of DNA
A.2.1 Deficiencies of the NBP reagent
A.2.2 Choice of reaction co-solvent
A.2.3 Influence of reaction mechanism and presence of scavengers
on shape of dose response curve
A.2.4 Mode of action of nitrogen mustard anticancer agents
|Christopher R. Lee was born in South Shields, England, in 1947. He received a BSc in Chemistry from the University of Sussex and a PhD in (biological) Psychiatry at the Medical Research Council Unit for Metabolic Studies in Psychiatry, University of Sheffield.
He stayed on at the MRC Unit, which had launched a project on periodic affective and schizo-affective disorders. He participated in the use of of gas chromatography-mass spectrometry for what are now called metabolomic studies, for quantitative analyses and for stable-isotopic tracer studies. This technique had become fairly routine but was still something of a technical challenge. The metabolic effects of lithium and rubidium administration were studied. A more ambitious project was to search for changes in the metabolic profile of patients during the mood swings of bipolar illness and other periodic disorders.
Later, he moved to the French pharmaceutical company Synthélabo, which subsequently merged with Sanofi, and eventually became Sanofi-Aventis. Projects, always with an analytical aspect (mainly chromatography, electrophoresis and mass spectrometry), were in the fields of discovery pharmacology, medicinal chemistry, metabolism and pharmacokinetics, and finally Chemistry, Manufacturing and Control (CMC). Some of the research in CMC was concerned with genotoxic impurities.