Evaluation of genotoxic risk of handling cytostatic drugs in clinical pharmacy practice.

The genotoxic risk of handling antineoplastic drugs was evaluated in fifteen women preparing chemotherapeutics in the Pharmacy Department of the University Hospital Maastricht. Twenty nurses of the same hospital, who were not exposed to cytostatics, acted as controls. Endogenous exposure to antineoplastic drugs was assessed by determination of urine mutagenicity, as well as by analysis of urinary methotrexate levels. As genotoxicological end-points, sister chromatid exchanges and hypoxanthine guanine phosphoribosyl transferase locus point mutations were studied in peripheral lymphocytes obtained via venous puncture. No differences in urine mutagenic activity, in sister chromatid exchange frequencies and in hypoxanthine guanine phosphoribosyl transferase point mutation frequencies between exposed and non-exposed groups were detected. Higher sister chromatid exchange frequency was observed in smokers as compared to non-smokers.

Immunochemical detection of rodent hepatic and urinary metabolites of cooking-induced food mutagens.

Monoclonal antibodies, previously selected to bind either 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) or 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) were evaluated to determine their binding properties with several known metabolites of these compounds purified from rat hepatocyte cultures. Both 2-N- and 5-substituted MeIQx metabolites were recognized by antibodies AIA-2 and AIA-11, while antibodies AIA-1 and AIA-12 bound N-substituted metabolites only. Anti-PhIP antibodies bound N-hydroxy-PhIP, N-sulfinamide-glutathione-PhIP and N-hydroxy-glucuronide-PhIP. Immunoaffinity columns incorporating these antibodies were used to concentrate and purify both the unmetabolized parent compounds and several relatively non-polar metabolites from the urine of rats exposed either to MeIQx or PhIP. Several of these metabolites corresponded with available standards of previously identified polar conjugate metabolites, e.g. N-sulfamate-MeIQx and N(OH)-gluc-PhIP, while others are as yet unidentified. Immunoaffinity chromatography is demonstrated as a promising means of selectively concentrating metabolites of PhIP and MeIQx from urine.

Formation of mutagenic urinary metabolites from 1-nitropyrene in germ-free and conventional rats: role of the gut flora.

1-Nitropyrene (NP), an environmental pollutant, a potent mutagen and an animal carcinogen, undergoes reduction, acetylation, ring-hydroxylation and conjugation in the rat in vivo to form mutagenic metabolites which are excreted in the urine. In order to investigate the role of the gut flora in the generation of these metabolites, germ-free rats of the AGUS strain, and conventional AGUS rats matched for sex and age, were injected i.p. with NP labelled with 14C. The germ-free rats excreted significantly less of the dose in urine than did the conventional rats. When urines were examined for mutagenicity with the Ames plate incorporation assay, the highest mutagenic activity was seen in the presence of S9 in 8-24 h urine from conventional rats. The conventional urines exceeded the germ-free urines by 10-fold in their content of 6-hydroxy-1-acetamidopyrene (NAAP-6-OH), previously identified as the predominant contributor to the mutagenicity of the urines of rats dosed with NP and excreted mainly as its beta-glucuronide conjugate. Conventional Charles River CD rats treated orally with D-glucaro-1,4-lactone, an inhibitor of beta-glucuronidase activity, excreted somewhat less NP-derived 14C in their urines over 48 h than did matched untreated rats, and their 8-24 h urines contained less than half as much of the mutagenic NAAP-6-OH as was found in the urines of the control rats. These results indicate that the gut flora are necessarily involved in the formation of NAAP-6-OH, and that both nitroreduction and the hydrolysis of glucuronides released for enterohepatic recirculation are essential in generating mutagenic metabolites from NP.

Excretion of mutagens following chemotherapy.

The mutagenic activity of urine was evaluated in children receiving single and multiple agent chemotherapy to determine the duration of carcinogenic risk to health care personnel and family contacts. Urine samples from 21 children were evaluated before and daily for 5 days after chemotherapy administration. Mutagenic activity, a sensitive though not specific indicator of carcinogenic risk, was assayed using mutant strains of Salmonella typhimurium (the "Ames test"). Validity of the assay was confirmed by demonstrating mutagenic activity in urine samples from 17 adult cigarette smokers but not from 21 adult nonsmokers (24/24 versus 0/37, p less than 0.001). None of the 21 children tested demonstrated mutagenic activity before chemotherapy administration. Following single agent dactinomycin, cyclophosphamide, daunorubicin, doxorubicin, methotrexate, or vincristine, mutagenic activity was demonstrated for 2 days (5/5 at 1 and 2 days and 0/5 at 3 days). Following multiple agent chemotherapy using two or three of the latter drugs on a single day, mutagenic activity was demonstrated for 4 or 5 days (16/16 at 1, 2, 3, and 4 days, and 4/16 at 5 days). Based on these observations with urine, and presumably other body fluids, precautions are recommended for 2 days following single agent and at least 5 days following multiple agent chemotherapy.

Correlations between urinary nicotine or cotinine and urinary mutagenicity in smokers on controlled diets.

Cigarette smokers have been reported to void urine that is more mutagenic, as measured in the Salmonella/microsome assay, than urine voided by nonsmokers. Several previous studies have attempted to correlate indices of tobacco smoke exposure (e.g. nicotine, cotinine, tar intake) with urinary mutagenicity, with conflicting results. These studies generally involved small numbers of smokers and did not carefully control diet, which is known to affect urinary mutagenicity markedly. Our objective was to conduct a controlled study to determine clearly if there were a correlation between urinary nicotine, cotinine, or nicotine + cotinine and urinary mutagenicity and to determine if nicotine or its major metabolite plays a role in the mutagenicity of urine from cigarette smokers. We used a large number of smokers (31), each of whom smoked both a tobacco-burning cigarette and a tobacco-heating cigarette on consecutive weeks, and we prepared and served identical diets to all subjects. Nicotine and cotinine concentrations were determined in small aliquots from urine samples collected over 24 hr, and the remaining urine sample was extracted and concentrated on XAD-2 resin for mutagenicity assays in the Salmonella/microsome test. Nicotine, cotinine, and nicotine + cotinine were statistically correlated with mutagenicity of urine from smokers of the tobacco-burning cigarette, but there was no correlation between nicotine, cotinine, or nicotine + cotinine and mutagenicity of urine from smokers of the tobacco-heating cigarettes. Thus, although urinary nicotine and cotinine concentrations correlate with urinary mutagenicity in smokers of tobacco-burning cigarettes, the present results indicate that nicotine and its metabolite are not responsible for the mutagenicity of smokers' urine.

Carcinogen hemoglobin adducts, urinary mutagenicity, and metabolic phenotype in active and passive cigarette smokers.

In 100 healthy volunteers, we have studied the relationship between the type (air- or flue-cured) and number of cigarettes smoked and different biomarkers relevant to the risk of bladder cancer, including the levels of 4-aminobiphenyl (ABP) hemoglobin adduct (a marker of internal dose), urinary mutagenicity in Salmonella typhimurium TA98, and the N-acetylation phenotype (a marker of susceptibility). ABP is a potent bladder carcinogen that is N-acetylated as an overall detoxification step. Levels of the ABP hemoglobin adduct were higher in smokers of black tobacco (air-cured) than in smokers of blond tobacco (flue-cured), confirming our earlier study. In addition, "slow" acetylators had higher levels of the ABP hemoglobin adduct for the same type and quantity of cigarettes smoked. Urinary mutagenicity was also associated with quantity of cigarettes but not with the acetylation phenotype. Convex dose-response relationships were found between the amount smoked and ABP hemoglobin adduct levels or urinary mutagenicity. In 15 nonsmokers who reported exposure to environmental tobacco smoke, ABP hemoglobin adduct levels, unlike urinary mutagenicity, were found to be an aspecific exposure indicator.

Markers of exposure to diesel exhaust in railroad workers.

Diesel exhaust is a known mutagen and a potential human carcinogen. Recent epidemiological studies have demonstrated a small increase in the risk of lung cancer from diesel exhaust exposure. However, many epidemiological studies have used crude estimates of exposure, and even accurate measures of exposure may not be accurate estimates of the internal dose received. Measurement of diesel exhaust exposure also has been limited by the absence of a standard marker. This study was undertaken to evaluate the usefulness of urinary mutagenicity as a biological marker of diesel exhaust exposure in the workplace. We measured the exposure of individual railroad workers to diesel exhaust by using personal air samples taken over two consecutive work shifts. Nicotine in the samples was measured to adjust the respirable particle concentrations for active and passive cigarette smoking. Urine samples were collected at the end of the study work shifts and were analyzed for markers of cigarette smoking (nicotine, cotinine) and for mutagenicity, using a sensitive microsuspension assay (micro preincubation assay; Salmonella strain TA98 with or without S9 enzyme). The number of cigarettes smoked on the study shift was recorded, and subjects completed a questionnaire at the end of the second day on personal habits and exposures at home and work. Multiple regression analyses were used to analyze independent determinants of urinary mutagenicity, including a generalized least-squares analysis that divided residual variation into between- and within-person components. Eighty-seven subjects completed 151 two-day protocols; an additional four subjects provided usable data for a single day (n = 306 samples). Respirable particle concentration was not a good marker of diesel exhaust exposure when contamination by environmental tobacco smoke existed in the work location, but respirable particle concentration that was adjusted for environmental tobacco smoke correlated with a priori assessments of diesel exhaust exposure by job grouping. Phenanthrene concentration, as a potential marker, was measured in a subset of personal samples, and correlated with known diesel exhaust exposure by job grouping. A constant ratio of phenanthrene to respirable particles in area samples from diesel exhaust-exposed work locations suggested that phenanthrene is promising as a marker for diesel exhaust. Mutagenic activity was also measured from extracts of respirable particles in a few personal filter samples, and this technique may be useful for further investigation in epidemiological studies.(ABSTRACT TRUNCATED AT 400 WORDS)

Gas chromatographic assay for N,N-dimethylglycine in urine.

A gas chromatographic method for the determination of N,N-dimethylglycine in urine has been developed. After clean-up by cation-exchange, N,N-dimethylglycine was derivatized with ethanol and hydrochloric acid to form the corresponding ethyl ester. After evaporation of solvent, N,N-dimethylglycine ethyl ester was extracted into methylene chloride and chromatographed on a gas chromatograph equipped with a packed column containing 10% Carbowax 20 M. The detection limit of the method is 0.01 mM N,N-dimethylglycine in urine. This method has been used to detect N,N-dimethylglycine in urine from healthy subjects as well as in urine from patients with metabolic disorders. These findings were verified by gas chromatography-mass spectrometry.

Effect of nutritional status on mutagenicity of urine excreted by rats treated with standard/experimental carcinogens.

Urine samples, collected from Sprague Dawley rats treated with extracts of tobacco/masheri, benzo (a) pyrene, N'-nitrosonornicotine, N'-nitrosodiethylamine and maintained on semi-synthetic diets sufficient or deficient in Vitamin A, B and protein were tested for mutagenicity using Salmonella/microsome assay. The mutagenic activity of urine or various treated groups was in the order deficient diet greater than standard laboratory diet greater than nutritionally sufficient diet. Present results confirmed the earlier observations that nutritionally deficient animals are likely to have more exposure to mutagenic metabolites that are generated by increased phase I enzymes and decreased detoxification system.

Clastogenic activity in urine of workers occupationally exposed to pesticides.

Genotoxicity in the urine of orchardists occupationally exposed to pesticides was investigated. Urine samples were obtained during pre-spraying and spraying periods from 22 non-smoking orchardists who spray large amounts of pesticides during the fruit growing season. For comparison purposes, urine was collected from 11 non-smoking personnel at an agricultural research station located near the application site and from 21 non-smoking individuals (reference controls) in a non-agricultural area. Organic material was isolated from urine by preparative reversed-phase high-pressure liquid chromatography, and assayed for clastogenic activity using Chinese hamster ovary cells. Urine samples collected during the pre-spraying period showed no significant differences in clastogenic activity compared to that found for the reference control group. However, clastogenic activity of urine specimens collected during the spraying period was significantly elevated (p less than 0.001) for the highly-exposed orchardists, but not for the research station personnel. Clastogenicity of orchardists' urine was observed within 8 h of pesticide application.

Determination of benzo[a]pyrene diol epoxide-DNA adducts in white blood cell DNA from coke-oven workers: the impact of smoking.

We have undertaken a study among coke-oven workers to test the feasibility of an enzyme-linked immunosorbent assay with anti-trans-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydro-benzo[a]pyrene- DNA antibodies for monitoring occupational exposure to polycyclic aromatic hydrocarbons (PAH). Coke-oven workers are occupationally exposed to relatively high levels of PAH and are at increased risk for lung cancer. Three blood samples were collected from each of the 56 coke-oven workers exposed to PAH and 44 unexposed workers employed in a steel-rolling factory of the same plant. In addition, PAH levels were measured in ambient air by personal sampling, and the excretion of 1-hydroxypyrene in urine was also measured on 3 consecutive working days. All participants were interviewed regarding working conditions, personal hygiene, and smoking habits. The results showed that the coke-oven workers were exposed to substantial concentrations of atmospheric PAH (1-186 micrograms/m3), including benzo[a]pyrene (0.1-7.8 micrograms/m3) and pyrene (0.6-23.6 micrograms/m3). Both benzo[a]pyrene and pyrene were shown to be representative for the whole group of PAH. Forty-seven percent of the coke-oven workers had detectable levels of PAH-DNA adducts in their white blood cells, compared with 30% of the controls. In both groups, smokers had significantly higher levels of PAH-DNA adducts than did nonsmokers. At one site, we found the correlation positive between DNA adducts and the duration of exposure (r = .47, P = .005). Generally, the correlation was not significant between PAH-DNA adducts in blood and the concentration of PAH in the air and 1-hydroxypyrene in urine.

Mutagenicity of deep-frying fat, and evaluation of urine mutagenicity after consumption of fried potatoes.

Mutagen formation during deep-frying was evaluated using standard frying conditions. Portions of pre-fried, sliced potatoes were fried in a commercial brand of hydrogenated vegetable frying fat, which was used repeatedly and for a prolonged period of time. Concentrations of polar oxidation and degradation products, and of dimeric and polymeric triglycerides, were found to increase in the frying fat as well as in fried potatoes with prolonged use of the fat. Thiobarbituric acid-reactive substances were detectable neither in the frying fat nor in the fried potatoes. Polar fractions of repeatedly used frying fat significantly increased the number of revertants in Salmonella typhimurium strain TA97 without S-9 mix. In the presence of S-9 mix mutagenic activity was reduced. As a consequence of ongoing formation of polar degradation and oxidation products, the mutagenicity of the fat increased after repeated use. Polar fractions of lipids extracted from commercially obtained pre-fried potatoes, as well as from fried potatoes, marginally increased the number of revertants in strain TA97 without S-9 mix. The mutagenicity of the lipid fractions of fried potatoes was not related to the heating time of the fat. Methanol extracts of fat-free residues of fried potatoes significantly increased numbers of revertants in strain TA97 after metabolic activation, which indicated that a different class of mutagens had been isolated. The mutagenicity of methanol extracts was not increased after either prolonged or repeated use of the fat. Urine samples of six healthy, non-smoking volunteers, collected during the 24 hr following consumption of portions of potatoes fried in repeatedly used fat, showed no increase in mutagenicity compared with control samples. Since the exact identity of mutagens formed during deep-frying, as well as their metabolic fate in man, is unclear at present, evaluation of possible adverse biological effects associated with consumption of fried foods will require strictly controlled metabolic studies.

Urine mutagenicity and biochemical parameters as markers of exposure to petroleum pitch using a rat model.

A petroleum pitch sample collected in a carbon electrode factory was studied using a series of in vivo assays for genotoxicity and enzymatic induction capability. Rats were treated with the petroleum derivative in three doses: 100, 50, and 10 mg/kg body weight. The treatment produced a rapid excretion of mutagenic substances in the urines of the first 24 hr only in rats treated with high doses (100 and 50 mg/kg). No faecal mutagenic activity was observed. Analyses of urinary thioethers showed that urinary metabolites derived from the compounds present in the pitch-sample at the lowest dose-administered (10 mg/kg) were eliminated primarily as cysteine conjugates. The pitch sample was found to be a good inducer of pulmonary and hepatic aryl hydrocarbon hydroxylase, especially after a 50 mg/kg dose. Urinary D-glucaric acid content was always statistically increased in treated animals compared with controls, confirming the enzymatic induction activity. Hepatic glutathione-S-transferase activity increased following treatment with 50 and 10 mg/kg doses.

Stability of the mutagenicity in stored cigarette smokers' urine and extract.

Urine from cigarette smokers was analyzed for the effect upon mutagenic activity when stored for as long as 175 days. Frozen aliquots of urine were thawed out at various time points in the study and prepared for bioassay. These urine extracts were not bioassayed immediately, but rather refrozen until all of the unprocessed urine samples had eventually been prepared for bioassay. All extracts were obtained using cyanopropyl solid phase extraction techniques. At the end of 175 days, all extracts were bioassayed using a microsuspension assay of Salmonella typhimurium TA98. Urine from smokers was found to be mutagenic (14.4-30.9 revertants/ml equivalent) while a control set of urine from non-smokers was not. Data from the storage study when analyzed by analysis of variance techniques indicated no statistical loss of mutagens occurred over the 175-day period although near significance was observed (P = 0.054). This near significance was the result of decreasing mutant response as storage time increased for two of the higher doses tested.

Genetic effects of benzene and radiation in ICR and X/Gf mice.

X/Gf mice (a tumor-resistant strain) were compared with ICR mice (moderately tumor-sensitive) for their sensitivity to chromosomal damage caused by benzene, cyclophosphamide (CP), benzo(a)pyrene (BP) and radiation. There was no difference between strains in the level of micronucleus formation caused by BP, CP or radiation. Although X/Gf mice metabolized somewhat less of the dose of benzene per weight than ICR mice, and had somewhat higher levels of genetic damage, it is not known whether X/Gf mice would be measurably more resistant to benzene carcinogenicity. Short-term genotoxicity tests are used as indicators of initiation, therefore, equal sensitivity to a set of standard clastogens suggests that tumor resistance in X/Gf mice is a function of later stages of carcinogenesis.

Is there influence of smoking on the mutagenicity of follicular fluid?

The mutagenicity of follicular fluid was examined in 24 patients, 12 smoking and 12 nonsmoking, who were treated in an in vitro fertilization program. The Salmonella microsome assay was used. It was found that the mutagenicity of follicular fluid was not influenced by the number of cigarettes smoked. Urine samples of smoking in vitro fertilization (IVF) patients however showed a dose-dependent elevation of the mutagenicity.

[The correlation between the excretion of thioethers and their mutagenic activity in urine in workers of different occupational groups]. / Untersuchungen über die Korrelation zwischen Ausscheidung von Thioethern und mutagener Aktivität im Urin bei Arbeitnehmern von verschiedenen Berufsgruppen.

Electrophilic substances can be inactivated by binding to glutathione or other SH-bearing molecules leading to urinary excretion of mercapturic acids or other thioether products. The mutagenic activity in urine as detected by mutagenicity assay (Ames-Test) is caused by genotoxic agents or their electrophilic metabolites. Therefore, it has been suggested that an effective protection by the glutathione system may diminish the urinary excretion of mutagens after exposure to genotoxicants. We determined the thioether concentration and mutagenic activity in urine samples of exposed workers (20 workers of a repair shop exposed to car exhaust, 35 workers of several dry cleaning shops exposed to halogenated hydrocarbons and 26 workers of a metal processing factory exposed to polycyclic aromatic hydrocarbons). We performed microfluctuation assay using Salmonella typhimurium TA98 and applied a method for the determination of urinary thioethers based on liquid chromatographic quantification of N-acetylcysteine. Our results show a linear correlation between the two exposure parameters which is independent on exposure conditions described above.

Daily use of smokeless tobacco: systemic effects.

STUDY OBJECTIVE: To compare exposure to nicotine and related cardiovascular effects as well as urinary mutagenicity (a potential marker of systemic absorption of carcinogenic compounds) during use of oral snuff, chewing tobacco, and cigarettes, as desired. DESIGN: Crossover sequential treatments, balanced-order experimental study. SETTING: Clinical research center. PARTICIPANTS: Eight healthy men who regularly smoked cigarettes and had previous experience with the use of both oral snuff and chewing tobacco. INTERVENTIONS: Four 3- or 4-day blocks during which participants used oral snuff, chewing tobacco, and cigarettes as desired, or abstained from all tobacco. Concentrations of nicotine and cotinine (the primary metabolite of nicotine), cardiovascular effects, and urine sodium, catecholamine and mutagenicity were measured over 24 hours at the end of each treatment block. MEASUREMENTS AND MAIN RESULTS: Circadian exposure to nicotine and cardiovascular effects, including urinary catecholamine excretion, were similar for all forms of tobacco use. Urine sodium excretion was greater while using smokeless tobacco than while smoking, probably due to absorption of sodium from the smokeless tobacco. Urine mutagenicity was markedly increased while smoking cigarettes and tended to be increased (P less than 0.10) while chewing tobacco but not while using oral snuff. CONCLUSIONS: Systemic absorption of nicotine, sodium, and carcinogenic chemicals from smokeless tobacco may cause or aggravate human illness in addition to the known adverse effects on the oral cavity.

Mutagens in urine of non-smoking and smoking workers in an aircraft tyre retreading plant. Skin exposure as a causal factor?

In an aircraft type retreading plant environmental samples taken at several departments showed mutagenic properties. Thursday urine samples of non-smoking and smoking workers showed higher urinary mutagenicity than urine samples collected on Sundays, thus suggesting occupational exposure to mutagenic substances. A relation between urinary mutagenicity on Thursdays and skin contamination measured on Wednesdays was observed. The data suggest that intake through the skin plays an important role in the occupational exposure to mutagenic compounds of rubber workers.