Urinary Mutagenicity Research Articles

Carcinogen Hemoglobin Adducts, Urinary Mutagenicity, and metabolic Phwnotype in Active and Passived Cigarette Smokers

Carcinogen Hemoglobin Adducts, Urinary Mutagenicity, and metabolic Phwnotype in Active and Passived Cigarette Smokers

The effect of exposure to nicotine, carbon monoxide, cigarette smoke or cigarette smoke condensate on the mutagenicity of rat urine

Cigarette smokers have been reported to void urine which is more mutagenic than that voided by non-smokers, but the specific urinary mutagen(s) have not been identified. Since mechanistic studies are best performed in animal models, the objective of this study was to determine if a model to study the role of cigarette smoke and its components in urinary mutagenicity could be developed in rats. XAD-2 resin was used to concentrate the urine and the microsuspension modification of the Ames test used to quantify mutagenicity. Nicotine administered by intraperitoneal injection at 0.8 mg/kg (the maximum tolerated dose) or inhalation of carbon monoxide for 14 days at the maximum tolerated dose (1800 ppm, resulting in 68% carboxyhemoglobin) did not increase urinary mutagenicity. Cigarette smoke condensate (CSC) prepared by electrostatic precipitation of mainstream smoke increased urinary mutagenicity at doses of 100 and 200 mg/kg when administered acutely by either i.p. injection or gavage, verifying that the assay system was capable of detecting cigarette smoke-related mutagens in the urine. However, cigarette smoke administered by the appropriate route of exposure, nose-only inhalation, for 1, 7, 14 or 90 days (1 h per day) did not increase urinary mutagenicity. The smoke concentration administered was at or near the maximum tolerated dose as evidenced by carboxyhemoglobin concentrations of approximately 50%, and of 10% or more weight loss in exposed animals. Thus, although cigarette smoke condensate is mutagenic in vitro and mutagenic urine was observed when rats were given high doses of CSC by inappropriate routes of administration, acute or subchronic inhalation exposure to the maximum tolerated dose of whole cigarette smoke did not increase urinary mutagenicity in rats. These results indicate that the rat may be an inappropriate model to study urinary mutagenicity following the inhalation of tobacco smoke.

Comparison of methods for the biomonitoring of nurses handling antitumor drugs.

Urinary mutagenicity, thioethers in urine, and sister chromatid exchanges and micronuclei in peripheral lymphocytes were determined for 60 nurses handling cytostatic drugs and 60 referents matched for sex, age, and smoking habits. Safety hoods were used by most of the nurses. The exposed nurses had more sister chromatid exchanges and higher urinary mutagenicity, as measured by Salmonella typhimurium TA 98, than the referents. There were no differences in the other tests. No dose-response relationship was established for any parameter. It was concluded that urinary mutagenicity with the Salmonella strain is the most sensitive test for monitoring nurses handling cytostatic drugs. Determining sister chromatid exchanges may also be a viable test, but it has the drawback of uncertainty as to whether the changes are attributable to present or past exposure. Only comparisons of rather large groups are useful, and a study design requiring matched referents would seem to be optimal.

Assessment of genotoxic exposure in Swedish coke-oven work by different methods of biological monitoring.

This study evaluated the results of several biological methods used simultaneously to monitor coke-oven work. Blood samples from 44 male coke-oven workers and 48 male referents, matched for age and smoking/snuff consumption, were examined for cytogenetic damage in lymphocytes. Urinary thioether excretion was determined for 62, and urine mutagenicity for 31, of the subjects, who followed a standardized diet during the urine sampling. Exposure to polycyclic aromatic hydrocarbons varied with work task, the ambient air levels of benzo[a]pyrene sometimes exceeding 5 micrograms/m3. Cytogenetic damage, urine mutagenicity, and thioether excretion did not differ between the groups. The smokers, however, had significantly higher sister chromatid exchange frequencies, urine mutagenicity, and thioether excretion than the nonsmokers. The absence of biological indications of genotoxic exposure was unexpected and indicates that the studied methods are not adequate to assess the carcinogenic risks of Swedish coke-oven workers.

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.

Role of bacterial nitroreductase and O-acetyltransferase in urine mutagenicity assay of rats exposed to 2,4,6-trinitrotoluene (TNT)

The urine mutagenicity of rats exposed to 2,4,6-trinitrotoluene (TNT) by i.p. injection was studied in the Salmonella assay using indicator strains with various levels of ‘classical’ nitroreductase or acetyl-CoA:Nrmhydroxylarylamine O-acetyltransferase activity. The strains used were the conventional Salmonella typhimurium TA98, nitroreductase-deficient TA98NR and -overproducing YG1021, and O-acetyltransferase-deficient TA98/1,8-DNP 6 and -overproducing YG1024. TA98, YG1021 and YG1024 clearly detected the increase of direct urine mutagenicity. A slight increase of mutagenicity was also detected with metabolic activation in YG1021 and YGI024. High levels of both nitroreductase and O-acetyltransferase significantly increased the sensitivity of the indicator strain to the mutagenicity of urine caused by TNT exposure, while the nitroreductase- or O-acetyltransferase-deficient strains gave negative responses.

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.

Monitoring the exposure of rats to benzo[a]pyrene by the determination of mutagenic activity in excreta, chromosome aberrations and sister chromatid exchanges in peripheral blood cells, and DNA adducts in peripheral blood lymphocytes and liver

The relative sensitivity of various methods suitable for monitoring the exposure to genotoxicants was studied in rats treated orally by gavage with benzo[a]pyrene (BP). The results were related to the occurrence of DNA adducts in liver. Male Wistar rats were treated once by gavage, BP doses ranging from 1 to 200 mg/kg body wt. The monitoring methods applied were: (i) mutagenicity in urine and extracts of faeces (Ames test, Salmonella typhimurium TA 100), (ii) chromosome aberrations (CA) and sister chromatid exchange (SCE) in peripheral blood lymphocytes (PBL), and (iii) DNA-adduct formation in PBL and liver (32P-postlabelling method). Mutagens in faeces and urine were detectable from dose levels of 1 and 10 mg/kg body wt respectively. Maximum mutagen levels were found in faeces (direct and indirect acting) collected 0-24 h after treatment and in urine (direct and indirect acting, glucuronidated and non-glucuronidated) collected 24-48 h after treatment. DNA-adduct formation was apparent in PBL at 10 mg (one spot) and 100 mg (two spots), and in liver at 100 mg/kg body wt (two spots). At the latter dose, the total BP-DNA adduct level in PBL was twice as high as in liver. The major adduct in PBL showed location and elution characteristics of the BP-adduct bound to N2 of doxyguanosine (BP-dG). None of the adducts in liver could be identified as BP-dG. A slight increase in SCE was seen in PBL only at 200 mg/kg body wt 6 h after treatment. CA did not increase at any of the dose levels used. Our results show that in vivo exposure of rats to BP can be detected by analysis of mutagenic activity in excreta and DNA-adducts in PBL. In contrast, measurement of CA and SCE in PBL appeared to be a rather insensitive method for detection of BP exposure.

Turmeric ( Curcuma longa)-induced reduction in urinary mutagens

Rats were fed turmeric at various levels in the diet for up to 3 months and then exposed to benzo[ a]pyrene (B[ a]P) or 3-methylcholanthrene (3-MC) by ip injection. Urinary mutagens were detected using the Salmonella typhimurium assay. Turmeric fed at 0.5% and above inhibited B[ a]P- and 3-MC-mediated mutagenicity. Turmeric did not adversely affect the food intake, or weight gain of the rats and no histological changes were detected. These findings are significant in view of the widespread exposure of humans to polycyclic aromatic hydrocarbons. The study has also revealed a useful in vivo model for testing the antimutagenicity.

32Postabelling analysis of urinary mutagens from smokers of black tobacco implicates 2-amino-1-methyl-6-phnylimidazo[4,5-b]pyridine (PhIP) as a major DNA-damaging agent

When mutagens extracted from the urine of two smokers of black tobacco were reacted with DNA in vitro in the presence of a metabolic activation system, several DNA adducts were detected by 32P-postlabelling analysis. Some of these adducts were also visible, but only faintly, on the autoradiogram for a non-smoker's urine. DNA adducts produced in vitro by 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline or 2-amino-1-methyl-6-phenylimidazo[3,5-b]pyridine could not account for the adduct pattern produced by the urinary mutagens. However, three or four 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-related DNA adducts were present among the five or six adducts observed for smokers in the autoradiograms of urinary mutagen-adducted nucleotides. Mutagenicity testing combined with HPLC fractionation of urinary extracts also supported the postlabelling data which implicates PhIP as a mutagen in the urine of smokers of black tobacco.

Effect of pentachlorophenol on the activation of 2,6‐dinitrotoluene to genotoxic urinary metabolites in CD‐1 mice: A comparison of GI enzyme activities and urine mutagenicity

2,6-Dinitrotoluene (2,6-DNT) and pentachlorophenol (PCP) are used for industrial purposes and are found in the environment as hazardous contaminants. Because concurrent exposure to both compounds can occur, it is of interest to determine if organochlorine compounds potentiate the effect of nitroaromatic chemicals. CD-1 mice were treated with PCP (42.8 mg/kg) for 4 weeks. On weeks 1, 2, and 4 after the initial PCP dose, mice were treated p.o. with 2,6-DNT (75 mg/kg) and 24 hr urines were collected. After concentration, the urines were tested for their mutagenic activity in Salmonella typhimurium strain TA98 without metabolic activation in a microsuspension bioassay. A significant increase (P less than .05) in mutagenicity was observed in urines from mice treated with 2,6-DNT alone and in combination with PCP. By week 4, mice that received both 2,6-DNT and PCP excreted urine that was more mutagenic than that from animals which received only 2,6-DNT. At weeks 2 and 4, mice were sacrificed and intestinal enzyme activities (nitroreductase, azo reductase, beta-glucuronidase, dechlorinase, and dehydrochlorinase) were quantitated. The enhanced genotoxicity observed in urines from 2,6-DNT/PCP-treated mice coincided with a decrease in nitroreductase and an increase in beta-glucuronidase activities in the small intestine.

Biological effects of short-term feeding to rats of repeatedly used deep-frying fats in relation to fat mutagen content

The effects of short-term feeding of mutagen containing, heated deep-frying oils on urinary and faecal mutagenicity, plasma clinical biochemical parameters, peroxidative effects and cell proliferative indices in the gastro-intestinal tract were determined in rats. Repeatedly used frying oils [a saturated fatty acid-rich coconut oil (CO) and a polyunsaturated fatty acid (PUFA)-rich (> 60% PUFA) vegetable frying oil (PO)] were administered to groups of seven rats at a level of 10% (by weight) in the diet for 4 wk; control groups were fed equal amounts of the unheated oils. Both heated oils showed direct-acting mutagenicity to Salmonella tester strain TA97; heated PO was also mutagenic to strain TA100. Both heated CO and heated PO contained detectable amounts of thiobarbituric acid-reactive substances (TBA-RS). In heated PO, hydroperoxides of linoleic acid were also present. In groups fed heated oils the mutagenicity of urine and faeces to strain TA97 was not found to be increased in comparison with the control groups. Faecal mutagenicity to strain TA100 was also unaffected by consumption of heated oils. Urinary excretion of TA100 mutagens was significantly increased in rats fed heated PO. Plasma alkaline phosphatase activity was clearly raised in rats fed heated PO, in comparison with rats fed unheated oils or heated CO. In addition, other clinical biochemical plasma parameters showed a tendency to be increased in rats fed heated PO, indicating hepatic and renal cellular toxicity. Urinary and faecal excretion of thiobarbituric acid-reactive substances (TBA-RS) were also slightly, but not significantly, increased in rats fed heated PO. Feeding heated CO to rats did not result in increased plasma enzyme activities and excretion of TBA-RS, nor in increased cell proliferation in gastro-intestinal tissues. Cell proliferation of the oesophageal tissues were slightly, but significantly, increased in rats fed heated PO, in comparison with the group fed unheated PO. Tissues of the glandular stomach and colon/rectum did not show significantly enhanced cell proliferation in the group fed heated PO. The results obtained in this study indicated that consumption of heated oils containing TA100 mutagens and oxidation products of linoleic acid produced indications of cellular damage to liver and kidneys, and increased urine mutagenicity, as well as enhanced cell proliferation in the oesophagus.

An experimental approach to identifying the genotoxic risk from cooked meat mutagens

In order to define the toxicological risk to the human population from the chemical compounds formed during the process of cooking animal meat, which have been described as possessing mutagenic, genotoxic and carcinogenic activities, an extensive study was undertaken of cooked meat extract and two cooked meat mutagens, 2-amino-3-methylimidazo(4,5- f)quinoline (IQ) and 2-amino-3,8-dimethylimidazo(4,5- f)quinoxaline (MeIQx). The study involved toxicokinetics and mouse-tissue distribution studies of the two chemicals, in vitro and in vivo mutagenicity/genotoxicity analyses (i.e. the detection of gene mutations, chromosome aberrations and micronuclei in mouse bone marrow cells, and mouse urine and faeces mutagenicity tests), as well as in vivo protein and DNA binding assays. IQ and MeIQx were found to be positive for the induction of gene mutations in Salmonella typhimurium TA98, but not in Chinese hamster V79 cells; IQ only was found to be positive for the induction of chromosome aberrations in Chinese hamster ovary cells and cultured human lymphocytes. IQ and MeIQx were negative for the induction of micronuclei in mice treated with 40 mg chemical/kg body weight; the lowest effective dose administered to the mice that produced mutagenic urine was 0.4 mg IQ/kg body weight and 0.04 mg MeIQx/kg. A dose of 40 mg IQ/kg, given orally by gavage to mice, produced an excretion of 1–4% of the applied dose in the urine and 0.1–2% of the applied dose in the faeces, when evaluated chemically or mutagenically. The number of DNA adducts in the liver correlated with the dose of IQ or MeIQx administered to the mice. All the data have been used for defining a possible risk estimate to the human population as a consequence of a cooked meat diet.

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.

The risks of handling cytotoxic drugs

Presuming that preparation of antineoplastic drugs without proper protection may lead to mutagenic urine (of which the effects are uncertain), one has to take great care when preparing these drugs. Apart from Norway no other country has national regulations issued by the government for handling cytostatic agents. Many organizations in various countries have made their own guidelines, which may be adapted to the situations in local hospitals. The following recommendations have been compiled after a review of the literature. They reflect a personal set of guidelines for preparation, administration and disposal of cytotoxic drugs. They are probably the minimum precautions that should be taken, and have not been approved by any committee or agency. Further precautions must first have their potential benefit weighed against probable inconvenience and additional costs.

Importance of exposure to gaseous and particulate phase components of tobacco smoke in active and passive smokers

The uptake of tobacco smoke constituents from gaseous and particulate phases of mainstream smoke (MS), inhaled by smokers, and of environmental tobacco smoke (ETS), breathed in by non-smokers, was investigated in two experimental studies. Tobacco smoke uptake was quantified by measuring carboxyhemoglobin (COHb), nicotine and cotinine in plasma and urine and the data obtained were correlated with urinary excretion of thioethers and of mutagenic activity. An increase in all biochemical parameters was observed in smokers inhaling the complete MS of 24 cigarettes during 8 h, whereas only an increase in COHb and, to a minor degree, in urinary thioethers was found after smoking the gas phase of MS under similar conditions. Exposure of non-smokers to the gaseous phase of ETS or to whole ETS at similar high concentrations for 8 h led to identical increases in COHb, plasma nicotine and cotinine as well as urinary excretion of nicotine and thioethers which were much lower than in smokers. Urinary mutagenicity was not found to be elevated under either ETS exposure condition. As shown by our results, the biomarkers most frequently used for uptake of tobacco smoke (nicotine and cotinine) indicate on the one hand the exposure to particulate phase constituents in smoking but on the other hand the exposure to gaseous phase constituents in passive smoking. Particle exposure during passive smoking seems to be low and a biomarker which indicates ETS particle exposure is as yet not available. These findings emphasize that risk extrapolations from active smoking to passive smoking which are based on cigarette equivalents or the use of one biomarker (e.g. cotinine) might be misleading.

Sensitivity of Salmonella typhimurium YG1024 to urine mutagenicity caused by cigarette smoking

Salmonella typhimurium YG1024, an O-acetyltransferase-overproducing derivative of TA98, was found to be more sensitive in detecting mutagenicity in human urine caused by cigarette smoking, than the conventional strain TA98, in Ames test in the presence of S9 mix. YG1021, another derivative of TA98 with elevated nitroreductase levels, did not show increased sensitivity. These results suggest that the mutagenic compounds concentrated by Blue Rayon from smokers' urine are aromatic amino compounds.

Chemical and Biological Studies of a Cigarette That Heats Rather Than Burns Tobacco

Cigarettes can be developed that heat rather than burn tobacco. Such products would be expected to have less "tar" and other combustion products than cigarettes that burn tobacco. With one product of this type, benzo(a)pyrene, N-nitrosamines, phenolic compounds, acetaldehyde, acrolein, hydrogen cyanide, and N-heterocyclic compounds have been reduced 10- to 100-fold compared to the Kentucky reference (1R4F) cigarette, a representative low-tar cigarette. The yields of nicotine and carbon monoxide from this new cigarette are less than the yields of 95% and 75%, respectively, of the cigarettes sold in the United States during 1988. Nicotine absorption from smoking this new cigarette is not significantly different from that of tobacco-burning cigarettes yielding equivalent levels of nicotine. The urine mutagenicity of smokers of new cigarettes is significantly less (P less than .05) than that of smokers of tobacco-burning cigarettes and is not significantly different (P greater than .10) from that of nonsmokers. We conclude that cigarettes which heat rather than burn tobacco can reduce the yield of tobacco combustion products. This simplification of smoke chemistry had no effect on nicotine absorption in smokers and resulted in a reduction of biological activity in smokers as measured by urine mutagenicity.

Comparative studies of the mutagenicity of urine from smokers and non-smokers on a controlled non-mutagenic diet

Measuring the mutagenicity of urine is widely viewed as a means of evaluating human exposure to potentially genotoxic materials. Diet and cigarette smoking have both been reported to affect the mutagenicity of human urine, but the relationship between smoking status and the expression of diet-related urinary mutagenicity is unknown. It has been reported that some promutagens are more active in in vitro assays when tested in the presence of urine from smokers than when tested in the presence of urine from non-smokers. We aimed to determine whether the differences in urinary mutagenicity between smokers and non-smokers result from increased urinary mutagenicity from dietary heterocyclic amine mutagens in smokers compared with non-smokers. Groups of smokers and non-smokers (6–12) were given identical diets, previously shown to be low in heterocyclic amines and very low in mutagenicity. The diet consisted exclusively of raw food and of food cooked in boiling water. After a 2-day dietary stabilization period, 24-hour urine samples were collected for three consecutive days. The regimen was repeated in the following week. For comparison, both groups were also placed on a “western” diet, consisting of a variety of foods prepared by several cooking methods, designed to reflect what a typical United States family might consume. Urine was concentrated using XAD-2 resin and then assayed for mutagenic activity in the Ames test. The urine of smokers was significantly more mutagenic than that of non-smokers when on both the raw/boiled and the “western” diets. These results indicate that the increased urinary mutagenicity observed in smokers compared with non-smokers is not due to enhanced mutagenicity of diet-related heterocyclic amine mutagens in the urine of smokers.

Urinary mutagens in municipal refuse incinerator workers and water treatment workers

Municipal refuse incineration workers may be exposed to mutagenic compounds from gaseous and particulate emissions and during ash removal operations. The frequency of urinary mutagens was measured by the Ames test among a sample of 104 refuse incinerator workers in seven incinerator plants during March-May 1988. The frequency was compared to that observed in 61 water treatment employees in 11 municipal water treatment facilities during the same period. Incinerator workers had a significantly higher risk for urinary mutagens and promutagens as compared to water plant workers after controlling for age. Among incinerator workers, increased risk of having urinary mutagens was associated with workers who wore protective clothing (defined as clothing other than masks or gloves) or whose job classification was equipment repair. It also showed a weak positive association with increasing age. There was an increased risk of urinary promutagens associated with not wearing gloves. The presence or absence of mutagenicity in workers' urine varied with plant location. Incinerator operating conditions affecting the production of toxicants and mutagens are discussed and the results of other studies involving toxicant exposure of humans near incinerators are cited.