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.

Decreased excretion of thioethers in urine of smokers after the use of beta-carotene.

Epidemiologic studies have demonstrated an inverse relation between vitamin A intake and lung cancer rate. There is strong evidence that the provitamin A, beta-carotene, plays a more important role in the protective effect than vitamin A itself. The anticarcinogenic properties of beta-carotene have so far been attributed to its scavenger properties in deactivating or trapping reactive chemical species such as singlet oxygen and certain organic free radicals. Smoking results in increased excretion of detoxification products of electrophilic agents (mercapturic acids) in urine. Since reactive electrophilic intermediates are involved in carcinogenesis, we performed a double-blind, placebo-controlled intervention trial to investigate whether the intake of beta-carotene by smokers would affect urinary thioether excretion. Before the intervention the beta-carotene group (n = 62) and the placebo group (n = 61) had similar thioether excretion levels in urine (4.2 vs 4.3 mmolSH/mol creatinine). During the intervention (20 mg beta-carotene daily for 14 weeks) the placebo group showed a 12% increase, whereas the beta-carotene group showed a 5% decrease (P = 0.004). After the intervention the beta-carotene group had a 15% lower thioether excretion level than the placebo group (4.1 vs 4.7 mmolSH/mol creatinine; P = 0.0017). Our study shows that urinary thioether excretion varies considerably over time, and that smokers have a decreased excretion of thioethers in urine after the use of beta-carotene.(ABSTRACT TRUNCATED AT 250 WORDS)

Mutagenicity of urine from rats after 1-nitropyrene and 2-nitrofluorene administration using new sensitive Salmonella typhimurium strains YG1012 and YG1024.

1-Nitropyrene (1-NP) and 2-nitrofluorene (2-NF), two of the most abundant nitro-substituted polycyclic aromatic hydrocarbons (nitro-PAH) present in combustion products such as diesel engine exhaust, were administered intraperitoneally to rats at a dose of 5 mg per animal. Urine samples, 1-NP and 2-NF were tested in the Ames assay using the newly developed Salmonella typhimurium strains YG1012 and YG1024 (overproducing O-acetyltransferase) and their parent strains TA1538 and TA98. In urine, collected over 3 periods of 24 h after administration, most of the mutagens appeared during the first 24 h. The mutagenicity was found to be a factor 2-30 higher in the YG strains when compared to the TA strains. Addition of S9 mix and rat liver cytosol both with and without beta-glucuronidase increased the mutagenicity of urine samples from 1-NP-treated rats. Addition of beta-glucuronidase revealed that a considerable part of the mutagenic metabolites of 1-NP and 2-NF were excreted as glucuronide conjugates. The increase in mutagenicity of urine samples from 2-NF-treated rats after the addition of rat liver cytosol referred to N,O-acyl transfer as a step in activating 2-NF to strong mutagens. The high sensitivity of the YG tester strains indicated that these strains might be used to explore environments where people are exposed to nitro-PAH, such as work places with diesel emission sources.

Non-mutagenicity of 4 metabolites of di(2-ethylhexyl)phthalate (DEHP) and 3 structurally related derivatives of di(2-ethylhexyl)adipate (DEHA) in the Salmonella mutagenicity assay.

Four metabolites of the rat liver carcinogen di(2-ethylhexyl)phthalate (DEHP) (mono-(2-ethylhexyl)phthalate, mono-(2-ethyl-5-hydroxyhexyl)phthalate, mono-(2-ethyl-5-oxohexyl)phthalate, and mono-(5-carboxy-2-ethylpentyl)phthalate) and 3 structurally related derivatives of di(2-ethylhexyl)adipate (DEHA) (mono-(2-ethylhexyl)adipate, mono-(2-ethyl-5-hydroxyhexyl)adipate, and mono-(2-ethyl-5-oxohexyl)adipate) were tested for mutagenicity in the Ames assay using Salmonella typhimurium strains TA97, TA98, TA100, and TA102, with and without a metabolic activation preparation. Aroclor 1254-induced rat liver S9 and DEHP-induced rat liver S9 were used. Concentrations of these compounds up to 1000 micrograms/plate were negative with all tester strains in the presence or absence of metabolic activation.

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.

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.

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.

1-Hydroxypyrene as an indicator of the mutagenicity of coal tar after activation with human liver preparations.

Liver S9 fractions were prepared from male Wistar rats, either non-induced or induced with Aroclor 1254 and from 5 human kidney transplant donors. The preparations were compared for their ability to metabolize the premutagens present in coal tar to mutagenic metabolites in the Salmonella mutagenicity assay towards strain TA98. Low levels of mutagenicity of coal tar were seen with human S9 preparations. The differences between the S9 mix of the 5 donors in capacity to activate premutagens were approximately 6-fold. The activation of coal tar by rat liver S9 preparations was higher than by the human S9 preparations. The metabolic conversion of pyrene in coal tar to 1-hydroxypyrene by the same human S9 preparations was determined in a parallel assay. 3 human preparations showed a high correlation between the formation of 1-hydroxypyrene and bioactivation of coal tar to mutagenic metabolites. The slope values of the individual regression lines were equal, suggesting that 1-hydroxypyrene is a good indicator for the activation of premutagens present in coal tar.

Mutagenicity of bi-, tri- and tetra-cyclic aromatic hydrocarbons in the "taped-plate assay" and in the conventional salmonella mutagenicity assay.

Aromatic hydrocarbons in the range of 1-4 nuclear rings were examined for mutagenicity in the so-called "taped-plate assay". This modification of the Ames assay is particularly equipped for the detection of volatile mutagens. Of the many compounds tested only phenanthrene, pyrene, benzo[c]phenanthrene and benzoacenaphthylene were positive in this assay. The present data underline the exceptional behaviour of fluoranthene by being a rather potent bacterial mutagen with a volatile nature (as found in a previous study).

Fluoranthene, a volatile mutagenic compound, present in creosote and coal tar.

Creosote, a coal-tar distillation product, contains mutagens which are volatile at 37 degrees C. After distillation of creosote we found that these volatile mutagens were present in the distillation fraction with the highest boiling range (greater than 360 degrees C). The "volatile mutagenic activity" was connected with the presence of fluoranthene, a polycyclic aromatic hydrocarbon. Commercially available fluoranthene was positive in the so-called "taped-plate assay" (the test system used for the detection of volatile mutagens) towards the strains TA98 and TA100 in the presence of S9 mix. The tested creosote and coal tar contained fluoranthene in concentrations of 5.2 and 2.2%, respectively.

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.

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.

Biological monitoring of polycyclic aromatic hydrocarbons. Metabolites in urine.

Assays of urinary mutagenicity, urinary 3-hydroxy-benzo(a)pyrene, and urinary 1-hydroxypyrene were used to study their suitability in estimating exposure to polycyclic aromatic hydrocarbons (PAH) in coal tar products. Rats exposed to coal tar solutions applied on the dorsal skin excreted mutagens, 3-hydroxy-benzo(a)pyrene, and 1-hydroxypyrene dose dependently in their urine. The correlation between the three parameters was high. Five dermatologic patients undergoing topical coal tar treatment excreted low concentrations of 3-hydroxy-benzo(a)pyrene and high concentrations of 1-hydroxypyrene. A significant correlation between the excretion of the two metabolites was found. The smoking workers of a coal tar distillation plant showed a significantly enhanced urinary mutagenicity compared with their nonsmoking colleagues, but an increase due to occupational exposure was not found. However, the concentration of 1-hydroxypyrene in the urine of these workers highly exceeded the upper 95 percentile of a reference population. The urinary excretion of 1-hydroxypyrene of smoking referents was not significantly increased compared with that of nonsmoking referents. The data suggest that urinary 1-hydroxypyrene is a sensitive and specific marker for the assessment of occupational exposure to PAH.

Excretion of 3-hydroxy-benzo(a)pyrene and mutagenicity in rat urine after exposure to benzo(a)pyrene.

3-hydroxy-benzo(a)pyrene (3-OH-B(a)P) and mutagenic activity in rat urine were determined after the oral administration of benzo(a)pyrene given in three repeated doses of 10, 20 and 50 mumol kg-1. The procedure for the determination of 3-OH-B(a)P consisted of enzymic hydrolysis, separation and HPLC-analysis. The mutagenic activity of concentrated urine samples was assayed with the Salmonella typhimurium strain TA98 in the presence of S9 mix and beta-glucuronidase. The urinary excretion of 3-OH-B(a)P and mutagens showed a correlation and both increased dose-dependently during the sampling period of 6 days. Data indicated that 3-OH-B(a)P can be regarded as a reliable representative of all urinary (pre)-mutagens derived from benzo(a)pyrene and exposure of rats to benzo(a)pyrene could be detected with greater sensitivity by the HPLC assay of 3-OH-B(a)P than with the non-specific mutagenicity assay.

Detection of volatile mutagens in creosote and coal tar.

Creosote and coal tar were examined for the presence of volatile mutagens by use of the so-called "taped plate assay". Application of this method, recently published by Distlerath et al., reveals that vapour escaping from creosote and coal tar at 37 degrees C was able to revert the Salmonella typhimurium strains TA98 and TA100 in the presence of S9 mix. The simplicity of this method makes it useful for routine screening of industrial fluids or solid products for the presence of volatile mutagens.

The presence of the mutagenic polycyclic aromatic hydrocarbons benzo[a]pyrene and benz[a]anthracene in creosote P1.

Several fractions of creosote P1 separated by TLC showed mutagenicity towards Salmonella typhimurium TA98. Thus mutagenicity is probably caused by the presence of mutagenic aromatic hydrocarbons. The mutagenic polycyclic aromatic hydrocarbons, benzo[a]pyrene and benz[a]anthracene, were detected in concentrations of 0.18 and 1.1% respectively. Because these compounds are probably not essential for the wood-preserving properties of creosote , a more selective composition of the product should be considered.

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.

Genotoxic exposure of workers creosoting wood.

In a small wood preserving industry spot samples were taken from contaminated surfaces at several places and tested for mutagenicity. The results suggest that the application of a wipe test can give a first indication of occupational exposure to mutagenic and carcinogenic substances, particularly when exposure occurs more from skin contact than from inhalation. One of the pesticide chemicals used to preserve wood is the mutagenic creosote . It was found that mutagens appeared in the urine of rats after intraperitoneal administration of creosote . Despite these results, no increase in mutagenicity could be detected in the urine of creosote workers in relation to their work.

Exposure to mutagenic aromatic hydrocarbons of workers creosoting wood.

Creosote P1 is mutagenic in the Salmonella microsome assay towards strains TA1537, TA1538, TA98 and TA100 in the presence of S9 mix. The mutagenic polycyclic aromatic hydrocarbons benzo[a]pyrene and benz[a]anthracene in this mixture are detected in concentrations of 0.18 and 1.1%, respectively. Spot samples taken from contaminated surfaces in several areas of a wood-preserving industry were tested for mutagenicity. The positive results suggest that a wipe test can give a first indication of occupational exposure to mutagenic substances, particularly when greater exposure occurs via skin contact than via inhalation. In urine of rats, mutagens appeared after treatment with creosote. However, no increase in mutagenicity could be detected in urine of creosote workers in relation to their work.