R. Colin Garner

Cyclophosphamide: Review of its mutagenicity for an assessment of potential germ cell risks

Cyclophosphamide (CP) is used to treat a wide range of neoplastic diseases as well as some non-malignant ones such as rheumatoid arthritis. It is also used as an immunosuppressive agent prior to organ transplantation. CP is, however, a known carcinogen in humans and produces secondary tumors. There is little absorption either orally or intravenously and 10% of the drug is excreted unchanged. CP is activated by hepatic mixed function oxidases and metabolites are delivered to neoplastic cells via the bloodstream. Phosphoramide mustard is thought to be the major anti-neoplastic metabolite of CP while acrolein, which is highly toxic and is produced in equimolar amounts, is thought to be responsible for most of the toxic side effects. DNA adducts have been formed after CP treatment in a variety of in vitro systems as well as in rats and mice using 3H-labeled CP. 32P-postlabeling techniques have also been used in mice. However, monitoring of adducts in humans has not yet been carried out. CP has also been shown to induce unscheduled DNA synthesis in a human cell line. CP has produced mutations in base-pair substituting strains of Salmonella tryphimurium in the presence of metabolic activation, but it has been shown to be negative in the E. coli chromotest. It has also been shown to be positive in Saccharomyces cerevisiae in D7 strain for many endpoints but negative in D62.M for aneuploidy/malsegregation. It has produced positive responses in Drosophila melanogaster for various endpoints and in Anopheles stephensi. In somatic cells, CP has been shown to produce gene mutations, chromosome aberrations, micronuclei and sister chromatid exchanges in a variety of cultured cells in the presence of metabolic activation as well as sister chromatid exchanges without metabolic activation. It has also produced chromosome damage and micronuclei in rats, mice and Chinese hamsters, and gene mutations in the mouse spot test and in the transgenic lacZ construct of Muta Mouse. Increases in chromosome damage and gene mutations have been found in the peripheral blood lymphocytes of nurses, pharmacists and female workers occupationally exposured to CP during its production or distribution. Chromosome aberrations, sister chromatid exchanges and gene mutations have been observed in somatic cells of patients treated therapeutically with CP. In general, there is a maximum dose and an optimum time for the detection of genetic effects because the toxicity associated with high doses of CP will affect cell division. In germ cells, CP has been shown to induce genetic damage in mice, rats and hamsters although the vast majority of such studies have used male mice.(ABSTRACT TRUNCATED AT 400 WORDS)

Use of microdosing to predict pharmacokinetics at the therapeutic dose: Experience with 5 drugs

A volunteer trial was performed to compare the pharmacokinetics of 5 drugs—warfarin, ZK253 (Schering), diazepam, midazolam, and erythromycin—when administered at a microdose or pharmacologic dose. Each compound was chosen to represent a situation in which prediction of pharmacokinetics from either animal or in vitro studies (or both) was or is likely to be problematic.

Testing of some azo dyes and their reduction products for mutagenicity using Salmonella typhimurium TA 1538.

A series of ten azo dyes as well as various single ring aromatic amines substituted on the benzene ring were tested for bacterial mutagenicity with Salmonella typhimurium TA 1538 using a soft-agar overlay method. Two dyes, sudan 2 and chrysoidin induced mutation but only in the presence of a rat liver preparation. Chrysoidin was the more active. Testing of its reduction products, aniline and 1,2,4-triaminobenzene showed a liver metabolite of the latter compound could be responsible for the mutagenic effect, having a comparable mutagenicity with 1,2-diamino-4-nitro-benzene, one of the mutagenic constituents of hair dyes. Structure-activity studies on a series of ring-substituted anilines indicated that mutagenic activity required at least two positions to be substituted with either amino or nitro groups, or one of each. The bacteria as well as the liver enzyme preparation may partake in the activation of these chemicals. The correlation between mutagenicity and carcinogenicity for this group of compounds is discussed.

Aflatoxin B-oxide generated by chemical or enzymic oxidation of aflatoxin B1 causes guanine substitution in nucleic acids

AFLATOXIN B1 (AFB1) has the highest biological activity of the four naturally occurring aflatoxins produced by the mould Aspergillus flavus1. Feeding studies have shown it to be the most potent liver carcinogen known for the rat2. Its ingestion by man is associated with the high primary liver cancer incidence in certain parts of Africa3. There is much evidence to suggest that AFB1 requires metabolism to exert both its carcinogenic and mutagenic effects (reviewed in ref. 4). Structure-activity5, as well as chemical studies6,7 indicate that the major route for activation proceeds through mixed function oxidase attack yielding the 8,9-oxide (previously called the 2,3-oxide but renumbered according to IUPAC recommendations). Attempts to chemically synthesise this metabolite have so far been unsuccessful. We report here that peracid oxidation of AFB1 generates AFB1-8,9-oxide, and that this latter compound reacts readily with nucleic acids to give adducts identical to those obtained after liver mixed function oxidase (MFO) activation in vitro and in vivo.

The role of DNA adducts in chemical carcinogenesis

The reaction of chemical carcinogens with DNA appears to be one of the earliest events in the initiation phase of cancer. These DNA reactions can be base- and position-specific, are affected by sequence context, and are repaired at different rates depending on whether or not they are on the transcribed or nontranscribed strand of DNA and which nucleotide sequence is modified. Thus, measurement of total genomic DNA reaction of carcinogens is only a crude first step in dissecting out which are the critical lesions for cancer initiation. On the other hand, we know that DNA adducts, which have been primarily characterised in experimental studies, appear to have similar structures in human DNA arising from occupational or environmental exposures. A number of different methods have been developed to detect and measure DNA adducts in man. These include physico-chemical methods such as mass spectrometry, 32P-postlabelling, fluorescence and accelerator mass spectrometry (AMS) and biological methods such as immunoassay. All these methods have their strengths and weaknesses. Human studies, using 32P-postlabelling, demonstrate that this method can be used to examine the effect of potential chemoprotective agents on DNA adduct level. AMS has been used to measure DNA adducts in human tissue after patients have ingested trace quantities of the food mutagens 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, a heterocyclic amine formed during the cooking of meat and the naturally occurring mycotoxin, aflatoxin B1. These studies can assist in assessing the risks associated with low-level exposure to food genotoxins.

The utility of microdosing over the past 5 years

Background: Microdosing studies (human Phase 0) are used to select drug candidates for Phase I clinical trials on the basis of their pharmacokinetic properties, using subpharmacologic doses (maximum 100 μg). There are questions as to whether pharmacokinetic data obtained at these low doses will predict those at the clinically relevant dose. Objective: To review the current literature on microdosing and assess how well microdose data have predicted the pharmacokinetics obtained at a therapeutic dose. Methods: All data published in the peer reviewed literature comparing pharmacokinetics at a microdose with a therapeutic dose were reviewed, excluding those studies aimed at imaging. Conclusions: Of the 18 drugs reported, 15 demonstrated linear pharmacokinetics within a factor of 2 between a microdose and a therapeutic dose. Therefore, data that support the utility of microdosing are beginning to emerge.

Macromolecular adduct formation and metabolism of heterocyclic amines in humans and rodents at low doses

2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are heterocyclic amines formed during the cooking of meat and fish. Both are genotoxic in a number of test systems and are carcinogenic in rats and mice. Human exposure to these compounds via dietary sources has been estimated to be under 1 microg/kg body wt. per day, although most laboratory animal studies have been conducted at doses in excess of 10 mg/kg body wt. per day. We are using accelerator mass spectrometry (AMS), a tool for measuring isotopes with attomole sensitivity, to study the dosimetry of protein and DNA adduct formation by low doses of MeIQx and PhIP in rodents and comparing the adduct levels to those formed in humans. The results of these studies show: 1, protein and DNA adduct levels in rodents are dose-dependent; 2, adduct levels in human tissues and blood are generally greater than in rodents administered equivalent doses; and 3, metabolite profiles differ substantially between humans and rodents for both MeIQx and PhIP, with more N-hydroxylation (bioactivation) and less ring oxidation (detoxification) in humans. These data suggest that rodent models do not accurately represent the human response to heterocyclic amine exposure.

MeIQx-DNA adduct formation in rodent and human tissues at low doses

Heterocyclic amines, such as 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), are mutagenic/carcinogenic compounds formed during the cooking of protein-rich foods. Human exposure to MeIQx has been estimated to range from ng/person/day to a few microgram/person/day. In contrast, animal studies have been conducted at doses in excess of 10 mg/kg/day. In order to determine the relevance of high-dose animal data for human exposure, the dose-response curves for [14C]-MeIQx have been determined in rodents at low doses under both single-dose and chronic dosing regimens using the high sensitivity of accelerator mass spectrometry (AMS). To make a direct species comparison, rodent and human colonic MeIQx-DNA adduct levels have been compared following oral administration of [14C]-MeIQx. The results of these studies show: (1) total MeIQx levels are highest in the liver > kidney > pancreas > intestine > blood; (2) MeIQx levels in the liver plateau after 7 days of chronic feeding; (3) hepatic MeIQx-DNA adducts begin to plateau after 2-4 weeks and reach steady-state levels between 4 and 12 weeks on chronic exposures; (4) hepatic DNA adducts generally increase as a linear function of administered dose for a single-dose exposure and as a power function for chronic feeding over a dose range spanning 4 orders of magnitude; (5) human colon DNA adduct levels are approximately 10 times greater than in rodents at the same dose and time point following exposure; and (6) > or = 90% of the MeIQx-DNA adduct in both rodent and human colon appears to be the dG-C8-MeIQx adduct. These studies show that MeIQx is readily available to the tissues for both humans and rodents and that adduct levels are generally linear with administered dose except at high chronic doses where adduct levels begin to plateau slightly. This plateau indicates that linear extrapolation from high-dose studies probably underestimates the amount of DNA damage present in the tissues following low dose. Further, if adducts represent the biologically effective dose, these data show that human colon may be as sensitive to the genotoxic effects of MeIQx as rat liver. The significance of these endpoints to tumor response remains to be determined.

Vegetable, fruit and meat consumption and potential risk modifying genes in relation to colorectal cancer.

Epidemiological evidence shows high red meat consumption to increase the risk of colorectal cancer, while the consumption of fruit and vegetables has been shown to be protective. Many genes have been identified that encode for enzymes involved in the metabolism of dietary carcinogens or anti‐carcinogens. A study of 500 incident colorectal cancer cases and population controls, matched for age, sex and general practitioner, was conducted in the United Kingdom to investigate whether 6 such genes (CYP1A1, GSTT1, GSTM1, GSTP1, EPHX1 and NQO1) modify the relationship between diet and disease risk. Usual diet was estimated using a detailed questionnaire administered by interview. Fruit and vegetable consumption were both found to protect against colorectal cancer, while overall meat and red meat consumption were found to increase risk. There was some evidence of interaction between GSTT1 and vegetable consumption (p=0.006, not adjusted for multiple tests) but no evidence of interaction with GSTM1. The protective effect of vegetables was only seen in those with deficient or intermediate GSTT1 predicted phenotype [OR 0.3, 95% confidence interval (0.1, 0.6), and OR 0.6 (0.4, 0.96), OR 1.4 (0.3, 2.4) for those with fast phenotype], and a similar result was observed for cruciferous vegetables. There was also weak evidence of interaction between red meat intake and GSTT1 (p=0.06), GSTP1 (p=0.16, with p=0.02 after adjustment for potential confounders) and NQO1 predicted phenotype (p=0.01). Because of the multiple hypotheses tested in our study, these findings require independent confirmation.

The use of isotopes in the determination of absolute bioavailability of drugs in humans

Absolute bioavailability studies in humans are not routinely performed as part of the drug registration process. They tend to be reasonably demanding, not least because toxicology data are required to support intravenous administration of a drug. Moreover, the classical crossover design of an absolute bioavailability study can suffer from artefacts caused by concentration-dependent pharmacokinetics. Many of the problems associated with absolute bioavailability studies can be alleviated using isotopically labelled drugs. Stable isotopes have been used in the performance of absolute bioavailability studies in humans for > 30years. More recently, the advantages of using radiolabelled drugs have been expanded by using the ultrasensitive technology of accelerator mass spectrometry. Isotopic labelling not only allows for the accurate and efficient determination of absolute bioavailability, but can also provide information on first-pass effects and other pharmacokinetic parameters.