Weekly patterns in smoking habits and influence on urinary cotinine and mutagenicity levels: confounding effect of nonsmoking policies in the workplace.

Lifestyle factors such as smoking have been shown to influence urinary mutagenicity. Therefore, these factors have to be considered carefully when evaluating occupational genotoxic exposures. We investigated day-to-day variability in active and passive tobacco smoke exposure by studying urinary cotinine levels and determined their influence on observed urinary mutagenicity. Urinary cotinine was assessed for 105 subjects employed in the rubber manufacturing industry in the Netherlands on Sunday, Wednesday, and Thursday. Urinary mutagenicity was measured by the Salmonella typhimurium strain YG1041 with metabolic activation for the Sunday urine sample and a pooled weekday urine sample. A sharp decrease in urinary cotinine concentration was observed during the week compared to Sunday for smokers (39%; P < 0.01) and nonsmokers (23%). Different smoking habits on Sunday resulted in higher regression coefficients for categorical proxies for smoking habits and urinary mutagenicity levels. However, regression coefficients for urinary cotinine and urinary mutagenicity were similar for the Sunday and weekday urine samples (beta = 0.29 and beta = 0.28, respectively). Consequently, these estimates were used to adjust urinary mutagenicity for tobacco smoke intake. Cotinine-adjusted urinary mutagenicity levels were comparable between smokers and nonsmokers, and a similar increase in urinary mutagenicity of 39% and 34%, respectively, was observed for both smokers and nonsmokers due to occupational genotoxic exposures or other changes in lifestyle factors. These results indicate that the introduction of nonsmoking policies in the workplace has reduced exposure to mainstream and environmental tobacco smoke, resulting in a temporal variation in lifestyle-related mutagenicity. Therefore, adequate adjustment for daily tobacco smoke exposure is a necessity when using the urinary mutagenicity assay to evaluate possible genotoxic exposures in the workplace.

Excretion of mutagens in human urine after passive smoking.

Eight non-smokers were experimentally exposed to cigarette smoke by staying in a poorly ventilated room together with heavy smokers for 6 h. Air samples were taken and the extract appeared to contain mutagenic substances. This is in accordance with the presence of carcinogens in tobacco smoke. Inhalation of the contaminated air by the passive-smokers resulted in an increase in the urinary excretion of products mutagenic in the Salmonella/microsome assay. This observation suggests that there is a causality in the association between increased cancer risk and passive-smoking, as was found by other investigators.

Influence of long-term ethanol treatment on in vitro biotransformation of benzo(a)pyrene in microsomes of the liver, lung and small intestine from male and female rats.

The influence of long-term ethanol exposure of rats on the microsomal biotransformation of benzo(a)pyrene [B(a)P] was studied. Male and female Wistar rats received an increasing amount of ethanol in their drinking water: percentages rose to 15% (w/v) in 3 weeks. The ethanol content was kept at a concentration of 15% for another 3 weeks. Livers, lungs and intestinal epithelial cells of the rats were then isolated and microsomal fractions prepared. In all organs, the metabolite most formed was 3-hydroxy-B(a)P. In the liver, males showed significantly higher B(a)P hydroxylase activity than females. On the basis of experiments using monoclonal antibodies, a significant part of the B(a)P biotransformation in male rat liver microsomes can be attributed to the male specific P4502C11. In the lung and intestine, there were no significant differences between the sexes. In the liver, ethanol treatment significantly decreased the microsomal formation of phenolic metabolites. In microsomes of intestinal epithelial cells, ethanol treatment enhanced the formation of phenols and diols. In conclusion, ethanol consumption by rats in moderate amounts leads to an alteration in the microsomal biotransformation of B(a)P. Effects are most prominent in the liver, where the formation of phenols is significantly decreased.

Nitroreduction and formation of hemoglobin adducts in rats with a human intestinal microflora.

In the covalent binding of nitroarenes to macromolecules, nitroreduction is an important step. The intestinal microflora represents an enormous potential of bacterial nitroreductase activity. As a consequence, the in vivo nitroreduction of orally administered nitroarenes is primarily located in the intestine. In this study, we have investigated the nitroreduction of 2-nitrofluorene (2-NF) by a human microflora in female Wistar rats. Germ-free (GF) rats were equipped with a bacterial flora derived from human feces. Nontreated GF rats and GF animals equipped with a conventional rat flora were used as controls. The composition of the human and the conventional microflora isolated from the rats were consistent with the microflora of the administered feces. In the rats receiving only sunflower seed oil, no adducts were detected. The animals equipped with a human or rat microflora that received 2-aminofluorene (2-AF) formed 2-AF hemoglobin (Hb)-adducts at average levels (mean +/- SEM) of 5.3 +/- 0.3 and 6.7 +/- 0.7 mumole/g Hb, respectively. After 2-NF administration, the adduct levels were 0.022 +/- 0.003 and 0.043 +/- 0.010 mumole/g Hb, respectively. In the GF rats, an adduct level of 0.57 +/- 0.09 was determined after 2-AF administration and no adducts were detected after 2-NF administration. The results show that nitroreduction by an acquired human intestinal microflora and subsequent adduct formation can be studied in the rat in vivo.

Drugs hazardous to healthcare workers. Evaluation of methods for monitoring occupational exposure to cytostatic drugs.

We review the literature concerning possible health risks for individuals (e.g. healthcare workers and pharmaceutical plant employees) occupationally exposed to cytostatic drugs. Cytostatic drugs possess toxic properties and may therefore cause mutagenic, carcinogenic and teratogenic effects. Hence, individuals handling these drugs in the course of their employment may face health risks. For this reason, it is important to monitor occupational exposure to these drugs. An overview of exposure monitoring methods is presented and their value is discussed. Most studies involve nonselective methods for biological monitoring and biological effect monitoring, such as the urinary mutagenicity assay and analysis of chromosomal aberrations and sister-chromatid exchanges in peripheral blood lymphocytes. The disadvantages of these biological methods are that their sensitivity is low and it cannot be proved beyond any doubt that the results found were caused by occupational exposure to cytostatic drugs. For occupational health services it is important to have sensitive and specific methods for monitoring exposure to cytostatic drugs. One of the most promising methods seems to be the determination of cyclophosphamide in urine using gas chromatography-tandem mass spectrometry. Several studies have demonstrated exposure to cyclophosphamide and other cytostatic drugs, even when protective measures were taken and safety guidelines were followed. To estimate the magnitude of any health effects arising from this exposure, we calculated the risk of cancer due to occupational exposure to cyclophosphamide on the basis of available human and animal dose-response data and the amounts of cyclophosphamide found in urine. The initial results show an extra cancer risk for pharmacy technicians and nurses.

1-Nitropyrene as a marker for the mutagenicity of diesel exhaust-derived particulate matter in workplace atmospheres.

The use of 1-nitropyrene (1-NP) as a marker for the occupational exposure to diesel exhaust (DE) mutagens was investigated in workplace atmospheres contaminated with DE from a variety of emission sources, such as power supplies, forklifts, trucks, caterpillar vehicles, trains, ships' engines, and vehicles in city traffic. Total suspended particulate matter was collected by area sampling. The 1-NP content of acetone extracts of these samples as determined by gas chromatography-high resolution mass spectrometry varied from 0.080 to 17 micrograms/g acetone extractable matter, corresponding to air concentrations of 0.012 to 1.2 ng/m3. A sample collected in a rural area contained 0.0017 ng/m3 1-NP. The mutagenicity of the extracts was tested in the Salmonella typhimurium strains TA98 and TA1538, using the microsuspension assay with and without metabolic activation by an exogeneous metabolizing system (rat liver S9-fraction). In addition, the S. typhimurium strains YG1021 and YG1024 were used because of their high sensitivity towards the mutagenicity of nitro polycyclic aromatic hydrocarbons. When plotting the mutagenic potency of the air sample extracts as determined in the absence of liver S9 versus the particle-associated 1-NP level, a relatively high correlation (r = 0.80-0.91) was observed in all of the S. typhimurium strains. High correlations (r = 0.80-0.93) were also observed when plotting the results of mutagenicity testing after activation by S9 versus the outcome of chemical analysis. These results show that the 1-NP content of workplace air samples is associated with their mutagenic potency, suggesting that 1-NP may be used as a marker for occupational exposure to DE-derived particle-associated mutagens.

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.

Appearance and reappearance of mutagens in urine from rats after oral administration of direct brown 95, due to coprophagy.

Rats treated orally with direct brown 95, a benzidine-based dye, widely used in dyeing of textiles, plastics, paper and other materials, showed 2 peaks of excretion of mutagenic products in urine, one between 6 h and 18 h after administration and one about 30 h later. Prevention of coprophagy by fitting neck collars resulted in the disappearance of the second peak. Oral administration of carminic acid resulted in a biphasic excretion of this dye in the feces, due to coprophagy. The excretion pattern of mutagens in urine after administration of direct brown 95 corresponds with the excretion pattern in the feces of orally administered carminic acid.

The appearance of mutagens in urine of rats after the administration of benzidine and some other aromatic amines.

The mutagenicity of urine from rats treated with benzidine or 5 other arylamines (0.25 mmol/kg; i.p.) was studied using the Ames-assay. It was found that samples of urine collected for 24 h after the administration of the carcinogens, benzidine, 4-aminobiphenyl and 2-aminonaphthalene, showed significantly mutagenic activity, whereas no mutagenicity was observed in urine after treatment with 3,3'-5,5'-tetramethylbenzidine, 2-aminobiphenyl and 1-aminonaphthalene. Mutagenic activities were dependent on the use of either hepatic S-9 Mix or cytosol as the activating enzyme preparation. The addition of beta-glucuronidase enhanced mutagenicity, except for 2-aminonaphthalene. The appearance of mutagens in urine was studied at varying doses of benzidine and at different time-intervals after the administration. The different excretion patterns found after the activation either with S-9 Mix or with cytosolic enzyme(s) suggest the presence in urine of different types of mutagenic products.

Studies on the metabolic activation of benzidine by isolated rat hepatocytes.

Isolated rat liver cells were shown to metabolize the aromatic amine benzidine to reactive products which are mutagenic to Salmonella typhimurium TA 1538 and which give rise to DNA excision repair within the liver cells. Intact rat liver cells are shown to be more active in the formation of mutagenic metabolites than the 9000-g supernatant from these cells. Data are presented which are in favour of the role of N-acetylation in this respect. Furthermore, indications are presented that a sulfation reaction is involved in the generation of DNA modifying metabolites, whereas formation of mutagenic products is likely to proceed via deacetylation and/or N,O-acyltransfer. Finally, data are given about the extrahepatocellular appearance of premutagenic metabolites which are more prone to metabolic activation by additional metabolic factors in the Salmonella assay than benzidine itself. The impact of these observations on the estimation of the genotoxic potential of benzidine will be discussed.

Metabolic activation of 2-aminofluorene by isolated rat liver cells through different pathways leading to hepatocellular DNA-repair and bacterial mutagenesis.

The aromatic amine 2-aminofluorene (2-AF) is metabolised by isolated rat liver cells to reactive species, thereby causing mutagenic effects in Salmonella typhimurium TA 1538 and evoking DNA-excision repair within the liver cells. The pathway leading to the production of metabolites mutagenic in Salmonella is likely to proceed via direct N-hydroxylation of 2-AF to N-hydroxy-2-aminofluorene (N-OH-2-AF). On the other hand, the formation of intermediates giving rise to hepatocellular DNA-repair is shown to depend upon N-acetylation of 2-AF to 2-acetylaminofluorene(2-AAF), whereas a subsequent conjugation reaction, most likely to be sulfate ester formation, is also essentially involved.

Involvement of non-oxidative enzymes in mutagenic activation of urine from rats, given benzidine and some other aromatic amines.

Urinary metabolites of rats treated with benzidine and some other genotoxic aromatic amines became mutagenic in the Ames assay after activation with liver cytosol from rat, mouse and guinea pig. Most of the mutagenic metabolites appeared in urine as glucuronides. Strong evidence was found that N,O-acyltransferase is responsible for the mutagenic activation by rat liver cytosol. The inhibitory effect of paraoxon and sodium fluoride indicates that the activation by mouse liver cytosol is due to the action of deacetylase. Mutagenic activation by guinea pig liver cytosol seemed to be mediated in part by deacetylase. The metabolite activated by these enzymes most likely is a glucuronidated, N-acetylated, N-hydroxylated product.

Metabolism of benzidine-based dyes and the appearance of mutagenic metabolites in urine of rats after oral or intraperitoneal administration.

The azo reduction and acetylation in vitro and the mutagenic activation in vivo of three azo dyes were studied. In the presence of rat-liver 9000 g supernatant benzidine was released from direct black 38 and direct brown 95, whereas hardly any benzidine was produced during incubation of direct blue 6. Incubation of benzidine with isolated rat hepatocytes resulted in the appearance of diacetylbenzidine. No diacetylbenzidine was formed during incubation of benzidine with rat-liver 9000 g supernatant, unless the cofactor for the acetylation reaction, acetyl coenzyme A, was added to the incubation medium. Isolated rat hepatocytes were capable to produce diacetylbenzidine from direct black 38, direct blue 6 or direct brown 95 without supplementation with acetyl coenzyme A. Administration of benzidine, direct black 38 or direct brown 95 to rats resulted in the appearance of mutagenicity in urine. For direct black 38 significantly higher mutagenicity values were found in urine after oral administration than after intraperitoneal treatment. Such differences were not observed for benzidine and direct brown 95. The results demonstrate that rat liver has a considerable capacity to reduce azo compounds. Nevertheless, for some compounds, like direct black 38, extrahepatic enzymes, most likely present in the intestinal flora, may also play a substantial role in the azo cleavage.

Internal exposure of rats to benzidine derived from orally administered benzidine-based dyes after intestinal azo reduction.

The role of the rat intestinal flora in the azo reduction of some benzidine-based dyes was studied in vitro and in vivo. The formation of benzidine was measured after anaerobic incubation of direct black 38, direct blue 6 and direct brown 95 in the presence of caecal bacteria in vitro. Benzidine was absorbed from the intestinal tract much better than the parent compounds. Oral administration of direct black 38 or direct brown 95 to Wistar rats results in the urinary excretion of mutagens. After oral administration of these dyes to germ-free Wistar rats no mutagenicity was observed in urine. The present results show that after oral administration, reduction by the intestinal flora should be considered as the first essential step in the biotoxification of benzidine-based dyes.

Excretion of benzo[a]pyrene and metabolites in urine and feces of rats: influence of route of administration, sex and long-term ethanol treatment.

The urinary and fecal excretion of benzo[a]pyrene (B[a]P) and its main metabolites were studied after oral and intraperitoneal administration of B[a]P to male and female ethanol-treated and non-ethanol-treated rats. After oral administration of B[a]P more mutagenic compounds as well as B[a]P metabolites were found in feces than after intraperitoneal administration. The excretion of B[a]P metabolites in urine and feces after oral administration were maximal at days 1 and 2 whereas after intraperitoneal administration excretion was maximal at days 2 and 3. In males, the amounts of excreted phenolic metabolites in urine and feces were generally higher than in females. The amounts of mutagenic products in urine and feces of males were also higher than in females after intraperitoneal and oral administration of B[a]P. In urine of female rats that received B[a]P intraperitoneally, a decreased excretion of phenolic metabolites was found after ethanol treatment. In feces of both male and female rats, a decreased excretion of 3-OH-B[a]P was found after ethanol treatment. In this study, the influence of sex and administration route on the excretion of B[a]P metabolites was more pronounced than the effect of ethanol treatment.

Influence of Aroclor 1254, phenobarbital, beta-naphthoflavone, and ethanol pretreatment on the biotransformation of cyclophosphamide in male and female rats.

The aim of the present study is to investigate the influence of the environmental factors, smoking and alcohol, on the biotransformation of cyclophosphamide (CP) in the rat in vivo and in vitro with S9 liver fractions. The biotransformation of CP was studied by the determination of the CP metabolites, nor-nitrogen mustard (NNM), 4-ketocyclophosphamide (KCP), and carboxyphosphamide (CAR). The effect of the environmental factors, smoking and alcohol consumption, on the biotransformation enzymes was mimicked by pretreatment of rats with beta-naphthoflavone and ethanol, respectively. Rats treated with olive oil and water served as controls and rats pretreated with Aroclor 1254 and phenobarbital were used as positive controls. The influence of sex and supplementation with NAD and GSH, mimicking a biological variation in NAD and GSH levels in rat and human liver, was also studied. Pretreatment of rats with Aroclor 1254 decreased the excretion of unmetabolized CP in urine, most likely due to an enhanced biotransformation. The in vitro hepatic biotransformation of CP in rats was strongly influenced by sex, by supplementation with NAD and GSH, and by pretreatment with the enzyme-inducers, phenobarbital and Aroclor 1254. No influence of pretreatment with the enzyme-inducers, beta-naphthoflavone and ethanol, was found. The results suggest that the influence of the environmental factors, alcohol consumption and smoking, on the biotransformation of CP in man will be negligible.

Biological fate of [14C]-1-nitropyrene in rats following intragastric administration.

1-Nitropyrene (1-NP), a weak carcinogen associated with diesel exhaust particles, has previously been detected in workplace atmospheres with in-use diesel engines and in the general environment. In order to gain insight in its biological fate, a single dose of [14C]-1-NP (27.6 microCi, 750 mg/kg body weight, b.w.) was administered intragastrically to rats and the presence of metabolites in blood and tissue homogenates, and radioactivity associated with blood proteins and tissue DNA, were studied. Early peak levels of radioactivity observed in blood and tissue homogenates indicated a rapid absorption of [14C]-1-NP from the gastrointestinal tract. Metabolite patterns observed in plasma, liver and kidney homogenates strongly suggested an important role of the intestinal microflora in the enterohepatic recirculation, but not in nitroreduction of 1-NP prior to absorption from the gastrointestinal tract. This might explain the low levels of radioactivity associated with blood proteins, since 1-nitrosopyrene, a product of nitroreduction of 1-NP, is likely to be involved in protein binding. Levels of radioactivity associated with plasma proteins were approximately four times higher than the levels of radioactivity associated with hemoglobin (401.0 and 84.1 pmol/g protein per micromol 1-NP kg b.w., respectively, at 24 h). Maximal 25% of the associated radioactivity was released following mild alkaline hydrolysis of either hemoglobin or plasma proteins. 1-Aminopyrene was the only released compound after hydrolysis of hemoglobin. In addition to 1-aminopyrene, two more polar unidentified metabolites were detected following hydrolysis of plasma proteins. Association of radioactivity with DNA was highest in the liver at the first moments of observation (7.4 pmol 14C Eq./mg DNA per micromol 1-NP kg b.w.), but decreased rapidly to levels lower than observed for kidney DNA (max. 3.0 pmol 14C Eq./mg DNA per micromol 1-NP kg b.w. at 24 h). In lungs 8-50 times less radioactivity was associated with DNA than observed in the liver and kidneys. The results of this study show, that 1-NP undergoes an extensive and complex biotransformation in vivo, resulting in a variety of metabolites present in blood and tissue homogenates and a diversity of blood protein adducts. Concentrations of plasma metabolites, blood protein adducts and DNA adducts were rather low. In addition, previous studies also showed relatively low concentrations of metabolites present in urine. Therefore, sensitive and selective methods will be needed in order to evaluate the biological fate of 1-NP, associated with diesel exhaust particles, in humans.

In vitro/in vivo effects of Mesna on the genotoxicity and toxicity of cyclophosphamide--a study aimed at clarifying the mechanism of Mesna's anticarcinogenic activity.

Cyclophosphamide is an effective antitumor agent with considerable side effects such as urotoxicity and carcinogenicity. These negative attributes may be caused by toxic and genotoxic metabolites, respectively. Mesna (sodium 2-mercaptoethane sulfonate) decreases the urotoxicity by scavenging the toxic metabolite acrolein. The present study was aimed at elucidating whether a similar scavenging of genotoxic alkylating intermediates could be found, which might cause the reduction in carcinogenicity. In vitro studies on the genotoxic and toxic properties of cyclophosphamide and its major metabolites to bacteria were therefore performed in the presence of Mesna. Mesna did not reduce the mutagenicity of any of the tested metabolites. Mesna clearly inhibited the toxic properties of acrolein. After in vivo application of Mesna and cyclophosphamide to rats, however, a lower yield of mutagens in the excreted urine was observed than after application of cyclophosphamide only.

Assessment of occupational exposure to diesel exhaust. The use of an immunoassay for the determination of urinary metabolites of nitroarenes and polycyclic aromatic hydrocarbons.

In a repair shop for train engines a pilot study was conducted to investigate occupational exposure to diesel exhaust. 1-Nitropyrene was determined in stationary sampled total suspended particulate matter collected on 2 consecutive workdays. Air concentrations of particulate associated 1-nitropyrene varied from non-detectable to 5.6 ng/m3. In spot urine samples collected on Sunday, Monday and Tuesday urinary metabolites of polycyclic aromatic hydrocarbons and their nitro-substituted derivatives were determined using an immunoassay. In the urine samples of 3 diesel mechanics both cumulative and average excretion of urinary metabolites over 48 and 72 h were significantly enhanced (P < 0.05) as compared to the excreted levels in urine samples from 2 office clerks.

Effect of airborne particles from selected indoor and outdoor environments on gap-junctional intercellular communication.

The effect of airborne particles from diesel exhaust, rubber and metal industry, urban air and biological sources (poultry, pig farming, compost industry) on gap-junctional intercellular communication (GJIC) were compared, using HEPA1c1c7 cells. Particles as such were compared with aqueous and organic extracts. Significant inhibition of GJIC by particle suspensions was only observed for the diesel and rubber samples, and for one biological sample (compost). Up to 83% of the inhibition of the whole suspension could be attributed to the particles as such. Washing the particles with organic solvents (aceton, methanol, hexane) did not result in a significant loss of activity from the particles, although the organic fractions showed a significant activity towards GJIC. More active organics was eluted from the rubber industry particles than from the diesel particles by the organic solvent. It is suggested that cancer promoting potential as measured by inhibition of GJIC may vary widely depending on the particle source, and that this effect may be exerted by the particles as such and/or by means of tightly bound bio-active material to the surface.