Mutagenicity of hexachlorobutadiene, perchlorobutenoic acid and perchlorobutenoic acid chloride

Hexachloro(1,3)butadiene (HCBD) is a well known environmental contaminant. The nephrocarcinogenic potential of HCBD has been shown in long-term studies with rats. Experiments were performed to assist in determining whether this effect is mediated by epigenetic or genotoxic mechanisms and to compare the mutagenic properties of HCBD with those of its monooxidation products, perchloro-3-butenoic acid (PCBA) and perchloro-3-butenoic acid chloride (PCBAC), which are conceivable metabolites of HCBD. All 3 compounds are mutagenic to the Salmonella typhimurium tester strain TA100. The mutagenic effect is dose-dependent and parallels the chemical reactivity of the compounds. HCBD is only mutagenic in the presence of drug-metabolizing enzymes (S9 mix) with an increased protein content. The mutagenic response after incubation with PCBAC and PCBA is 2-3-fold that of HCBD. Additionally, both PCBAC and PCBA exert a mutagenic response in the absence of S9 mix. The experiments support the assumption of a genotoxic potential of HCBD.     

An Ecological Risk Assessment of Hexachlorobutadiene

Hexachlorobutadiene (HCBD) has never been commercially produced in Canada and was imported in the past for use as a solvent. Anthropogenic activity is linked with the entry of this substance into the environment. While current Canadian sources of HCBD involve low-level releases, potentially they can be numerous. Until recently, the most significant point source of HCBD in Canada appeared to be the Cole Drain, which discharges into the St. Clair River at Sarnia, Ontario, and includes outfalls from an industrial landfill and a few industrial companies. HCBD has been detected in Canadian surface waters, sediments, aquatic organisms and, occasionally, air. Considering the properties of the substance, including its persistence and bioaccumulation characteristics, the environmental risk assessment of HCBD was focused on the aquatic environment. The results of a conservative assessment suggest that there is a risk of harmful effects for benthic organisms exposed to sediments contaminated by HCBD in the most contaminated part of the St. Clair River.     

Cyanocobalamin Enables Activated Sludge Bacteria to Dechlorinate Hexachloro-1,3-butadiene to Nonchlorinated Gases

The ability of activated sludge obtained from a local wastewater treatment plant to dechlorinate hexachloro-1,3-butadiene (HCBD) in the presence of either acetate or lactate and cyanocobalamin was investigated. Results from headspace analysis indicated complete dechlorination of HCBD by the accumulation of fully dechlorinated C4 gases (1-buten-3-yne, 1,3-butadiene, and 1,3-butadiyne). Dechlorination products were not observed in the control cultures without cyanocobalamin. Examination of control cultures revealed that the disappearance of HCBD from the headspace was partly due to adsorption into the biomass. However, the key for dechlorination was the shuttle (cyanocobalamin) rather than specific microbial enzymatic activity. The hypothesis that the bacteria reduced cyanocobalamin, which in turn reductively dechlorinated HCBD, was supported by the finding that cyanocobalamin reduced by zero-valent zinc resulted in complete dechlorination. The significance of the findings is that, in contrast to prior work where specific anaerobic bacteria (enrichments or pure cultures) were believed to be necessary for dechlorination resulting in only partly dechlorinated products, the currect data show that nonspecific aerobic activated sludge bacteria can be employed for complete HCBD dechlorination at rates sufficiently high to be considered for bioremediation projects.     

Options for In Situ Remediation of Soil Contaminated with a Mixture of Perchlorinated Compounds

Environments contaminated with mixtures of chlorinated hydrocarbons represent a formidable challenge for bioremediation because biodegradation of all components of the mixture must be demonstrated. In this study a soil site contaminated with hexachloro-1,3-butadiene (HCBD), hexachlorobenzene (HCB), and perchloroethene (PCE) was investigated. Environmental parameters (including toxicity) and microbial community composition were characterized. The lack of scientific literature on HCBD biodegradation led to attempts to develop HCBD-respiring enrichment cultures and to test the hypothesis that known PCE-degrading cultures could dechlorinate HCBD. No HCBD dechlorination was observed. An alternative approach, using electron shuttles to degrade the mixture of chlorinated hydrocarbons, was compared with the activity of zero-valent iron. The authors conclude that electron shuttles offer promise for the in situ treatment of mixtures of chlorinated hydrocarbons.     

六氯-1,3-丁二烯的微生物降解研究进展

六氯-1,3-丁二烯(hexachlorobutadiene,HCBD)是一种有毒有害的脂肪族氯代烃,曾经作为杀虫剂、除草剂、变压器油和传热流体等化学工业产品的重要成分被广泛应用于生产生活。HCBD因满足《关于持久性有机污染物的斯德哥尔摩公约》中风险筛选标准(如毒性、持久性、远距离环境迁移和生物累积性等),缔约方于2015年第七次会议中将其增列为持久性有机污染物,2017年又将其列入该公约的附件Ｃ以控制其环境排放量。目前关于HCBD的环境归趋仍是研究热点,但是对于HCBD的微生物降解转化机制尚缺乏深入研究。鉴于此,本文重点回顾并讨论了地下水、底泥等厌氧环境中已报道的HCBD微生物降解转化途径、速率及机制,并从热力学角度阐述HCBD及其降解产物作为电子受体通过还原性脱氯反应被厌氧脱卤微生物代谢转化的可行性。最后,本文根据现有研究结果,提出微生物厌氧降解HCBD的研究展望,包括多组学技术解析HCBD降解功能菌群结构和潜在互作机制、HCBD厌氧降解微生物的分离与纯化,以及HCBD厌氧降解菌剂的开发与污染场地原位生物修复应用等。     

Identification and degradation characterization of hexachlorobutadiene degrading strain Serratia marcescens HL1

A bacterium (strain HL1) capable of growing with hexachlorobutadiene (HCBD) as sole carbon and energy sources was isolated from a mixture of soil contaminated with HCBD and activated sludge obtained from petrochemical plant wastewater treatment plant by using enrichment culture. Biochemical characteristics and phylogenetic analysis based on 16S rDNA sequence indicate that strain HL1 clearly belongs to Serratia marcescens sp. Resting cells of strain HL1 were found to remove HCBD from culture fluids with the concomitant release of chloride ion under aerobic conditions. The ranges of pH value and temperature for satisfactory growth of strain HL1 cells were from 7.0 to 8.0 and 25 to 30 degrees C, respectively. Capability of resting cells to degrade HCBD was induced by HCBD in the culture fluids. HCBD (20mg/l) was removed from culture fluids by resting cells in 4 d without lag phase, but for 50mg/l and 80mg/l HCBD 7 days were needed with lag phase. Growth of strain HL1 cells was inhibited by HCBD at the concentration up to 160mg/l. First order kinetics could be fitted to the biodegradation of HCBD by HL1 cells after lag phase at initial concentrations of 20, 50, and 80mg/l. Strain HL1 also showed strong capacity to degrade chloroprene, trichloroethylene, tetrachloroethylene, and vinyl chloride at solely initial concentration of 50mg/l. Results could offer useful information for the application of strain HL1 in bioremediation or control of HCBD-polluted environment.     

Effects of age and sex on hexachloro-1,3-butadiene toxicity in the fischer 344 rat

Effects of age and sex on hexachloro-1,3-butadiene (HCBD) nephrotoxicity were determined 24 hours after a single dose (0, 25, 50, 100 or 200 mg/kg) in 28- and 63-day-old Fischer 344 rats. HCBD treatment significantly increased the kidney to body weight ratio but had little effect on the liver to body weight ratio. The 28-day-old rats were more susceptible to HCBD nephrotoxicity judged by elevated blood urea nitrogen, decreased renal cortical accumulation of p-aminohippurate tetraethylammonium. Adult female rats (63-day-old) appeared to be more susceptible to HCBD nephrotoxicity than males at the low dose (50 mg/kg).     

Development of resistance against hexachlorobutadiene in the proximal tubules of young male rat

Hexachlorobutadiene (HCBD) is a potent nephrotoxin in rodents that can cause degeneration, necrosis and regeneration in renal tubular epithelial cells. Its toxicity is due to its conjugation by glutathione (GSH) to form glutathione S-conjugate, by the enzyme glutathione S-transferase and finally to the related cysteine-conjugate. This metabolite is then actively taken up by kidney and cleared in the renal tubular epithelial cells, rich in beta-lyase, to a reactive thiol derivative that covalently binds to the macromolecules. In this study, different groups of 28-day male Wistar albino (W/A) rats were dosed daily with 25 mg/kg HCBD for 2, 3, 4 and 7 days; control group dosed with corn oil. Data showed that in the 2- and 3-day treated groups there was substantial necrosis to the straight portion of the proximal tubules (pars recta or S3 segment), rich in glutamine transaminase K (GTK/beta-lyase). In the 4-day treated group, the renal proximal tubules had regenerated and showed a basophilic appearance. In animals treated for 7 days, it was observed that the kidney appeared to have returned to normal and had become resistant to further doses of HCBD. To define the time for the kidney to regain susceptibility to HCBD, 18- and 25-day studies with both low (25 mg/kg) and high (100 mg/kg) doses of HCBD (following two initial doses of 25 mg/kg) were performed. In the 18-day study, histopathological examination of the kidneys in animals of this group and also animals in the 25-day study, which received two further doses of HCBD, showed that the severity of kidney damage is much less than in the 2-day treated animals, a clear indication that the tubular cells were still resistant to the low dose of HCBD. Concentration of blood urea nitrogen, as a marker of kidney damage, in these two groups also confirmed the results. In animals re-exposed to the high dose of HCBD, data showed that the susceptibility to HCBD was starting to return.     

Bacterial beta-lyase mediated cleavage and mutagenicity of cysteine conjugates derived from the nephrocarcinogenic alkenes trichloroethylene, tetrachloroethylene and hexachlorobutadiene

The metabolism of beta-lyase and the mutagenicity of the synthetic cysteine conjugates S-1,2-dichlorovinylcysteine (DCVC), S-1,2,2-trichlorovinylcysteine (TCVC), S-1,2,3,4,4-pentachlorobuta-1,3-dienylcysteine (PCBC) and S-3-chloropropenylcysteine (CPC) were investigated in Salmonella typhimurium strains TA100, TA2638 and TA98. The bacteria contained significantly higher concentrations of beta-lyase than mammalian subcellular fractions. Bacterial 100,000 X g supernatants cleaved benzthiazolylcysteine to equimolar amounts of mercaptobenzthiazole and pyruvate. DCVC, TCVC and PCBC produced a linear time-dependent increase in pyruvate formation when incubated with bacterial 100,000 X g supernatants; pyruvate formation was inhibited by the beta-lyase inhibitor aminooxyacetic acid (AOAA). CPC was not cleaved by bacterial enzymes to pyruvate. DCVC, TCVC and PCBC were mutagenic in three strains of S. typhimurium (TA100, TA2638 and TA98) in the Ames-test without addition of mammalian subcellular fractions; their mutagenicity was decreased by the addition of AOAA to the preincubation mixture. CPC was not mutagenic in any of the strains of bacteria tested. These results indicate that beta-lyase plays a key role in the metabolism and mutagenicity of haloalkenylcysteines when tested in S. typhimurium systems. The demonstrated formation in mammals of the mutagens DCVC, TCVC and PCBC during biotransformation of trichloroethylene (Tri), tetrachloroethylene (Tetra) and hexachlorobutadiene (HCBD) may provide a molecular explanation for the nephrocarcinogenicity of these compounds.     

Urinary markers of nephrotoxicity following administration of 2 bromoethanamine hydrobromide a comparison with hexachlorobutadiene

2 bromoethanamine hydrobromide (BEA) has been widely considered to be a target selective nephrotoxin that causes necrosis of the medulla in 24-48 h, but recent reports suggest that early cortical injury is also associated with this lesion. In order to assess the cortical effects of BEA (100 mg kg^-1 bw single ip injection), several urinary markers of renal injury were evaluated over a 7 day period in male Wistar Albino rats. Hexachlorobutadiene (HCBD 150 mg kg^-1 bw in peanut oil ip), a renal toxin which targets selectively for the proximal tubule, was used as a comparison. After BEA treatment, urinary levels of alanine aminopeptidase, gamma-glutamyl-transpeptidase, alkaline phosphatase and glucose increased transiently. Each of the proximal tubule marker enzymes peaked earlier following HCBD treatment and elevation of alanine aminopeptidase and gamma glutamyl transpeptidase was sustained for longer periods than for BEA. Following BEA treatment, lactate dehydrogenase rose prominently on day 1 followed by a return to control values on day 2 and a further rise on day 3 and remained high until the end of the study. BEA also increased the urinary excretion of total protein and albumin. After HCBD treatment, lactate dehydrogenase showed a transient elevation and glucose levels were slightly increased. Based on the present observations the changes induced by BEA administration on urinary markers of renal injury are different from those observed following HCBD treatment. These findings suggest that BEA toxicity also involves other parts of the kidney besides the papilla.     

Urinary markers of nephrotoxicity following administration of 2 bromoethanamine hydrobromide a comparison with hexachlorobutadiene

2 bromoethanamine hydrobromide (BEA) has been widely considered to be a target selective nephrotoxin that causes necrosis of the medulla in 24-48 h, but recent reports suggest that early cortical injury is also associated with this lesion. In order to assess the cortical effects of BEA (100 mg kg^-1 bw single ip injection), several urinary markers of renal injury were evaluated over a 7 day period in male Wistar Albino rats. Hexachlorobutadiene (HCBD 150 mg kg^-1 bw in peanut oil ip), a renal toxin which targets selectively for the proximal tubule, was used as a comparison. After BEA treatment, urinary levels of alanine aminopeptidase, gamma-glutamyl-transpeptidase, alkaline phosphatase and glucose increased transiently. Each of the proximal tubule marker enzymes peaked earlier following HCBD treatment and elevation of alanine aminopeptidase and gamma glutamyl transpeptidase was sustained for longer periods than for BEA. Following BEA treatment, lactate dehydrogenase rose prominently on day 1 followed by a return to control values on day 2 and a further rise on day 3 and remained high until the end of the study. BEA also increased the urinary excretion of total protein and albumin. After HCBD treatment, lactate dehydrogenase showed a transient elevation and glucose levels were slightly increased. Based on the present observations the changes induced by BEA administration on urinary markers of renal injury are different from those observed following HCBD treatment. These findings suggest that BEA toxicity also involves other parts of the kidney besides the papilla.     

Mutagenic activity and structure-activity relationships of short-chain dialkyl N-nitrosamines in a hamster hepatocyte V79 cell-mediated system

A series of 19 short chain dialkyl N-nitrosamines was studied for mutagenic activity in an uninduced hamster hepatocyte V79 cell-mediated mutagenesis system. Ouabain was used as the selective agent to quantitatively analyze for chemically induced mutants. None of the nitrosamines was mutagenic in the absence of hamster hepatocyte activation. The relative mutagenic activities of the nitrosamines at an equimolar dose are presented. The results of the study indicated that: (a) increasing alkyl chain length decreased mutagenic activity; (b) oxidation of the carbon position to a carbonyl group increased the mutagenic activity of symmetrical and asymmetrical nitrosamines, whereas oxidation to a hydroxyl group only increased the mutagenic activity of the asymmetrical nitrosamines tested and (c) the carbon position at which oxidation occurred was important in determining mutagenic activity. The relationships between structure, metabolic activation, and mechanisms of mutagenic activity are discussed.     

Mutagenic profile of rubber dust and fume exposure in two rubber tire companies

The aim of this study was to evaluate current mutagenic activity of ambient rubber dust and fume exposure in the mixing and curing departments of two rubber tire companies situated in The Netherlands and Sweden. Salmonella typhimurium strains YG1021, YG1024 and YG1041 were used to study the possible presence of mutagenic nitroarenes and aromatic amines. A large difference in mutagenic activity was found between the two companies. While the rubber tire company situated in The Netherlands revealed overall high mutagenic activity of rubber dust and fumes in the mixing and curing departments, respectively, 430 and 279 rev/m(3) (YG1041), the Swedish company showed almost no mutagenic activity, respectively, 18 and 54 rev/m(3) (YG1041). Further identification of the mutagenic profile showed that mutagenic activity was exclusively observed in S. typhimurium strains with elevated levels of O-acetyltransferase activity (YG1041 and YG1024) in the presence of a metabolic active liver S9 fraction, possibly indicating the presence of indirect mutagenic aromatic amines. These results show that although production processes and lay-out within rubber tire companies are comparable, differences in rubber chemicals used and overall level of control measures (e.g., good housekeeping, cleanliness) are likely to result in substantial differences in mutagenic exposure levels between companies.     

The release of mutagens from airborne particles in the presence of physiological fluids

Airborne particulates collected indoors in residences and outdoors were extracted by soxhlet extraction and sonication with methanol. In a comparative study in which mutagenic activity was evaluated in the Salmonella typhimurium reversion assay both soxhlet extraction and sonication proved to be suitable extraction methods. First, the residue, obtained by sonication of loaded filters with methanol followed by evaporation to dryness (tar), was sonicated with newborn calf serum and lung lavage fluid from pigs. All serum extracts of the tar were mutagenic to Salmonella typhimurium TA98, and contained direct- and indirect-acting mutagens. However, the mutagenic activity recovered by serum was only about half of the total mutagenic activity of the tar. The other part of the mutagenic activity remained in the tar. Lung lavage fluid was only able to remove 5-10% of direct-acting mutagens from the tar of all samples. About 20% of indirect-acting mutagens from indoor air were recovered in lung lavage fluid, while the lung lavage fluid extract from outdoor air did not show indirect mutagenic activity. Second, mutagenic activity recovered by direct sonication of the filters with physiological fluids was comparable with the recovery obtained by sonication of the tar. However, after sonication of the filter with lung lavage fluid hardly any mutagenic activity remained on the filter, whereas after sonication of the tar a clear mutagenic activity was observed in the non-soluble residue.     

Unchanged iron and copper and increased zinc in the blood of obese children after two hypocaloric diets

Serum iron (sFe), and ferritin (sFert), transferrin saturation index (TSI), plasma zinc and copper (pZn, pCu), and erythrocyte zinc content (eZn) were measured in 55 obese children and adolescents (28 males and 27 females) before and after a 13-wk treatment with a hypocaloric balanced diet (HCBD, 22 subjects) or a 10-wk treatment with a protein sparing modified fast diet (PSMF, 33 subjects). The energy intake provided by the HCBD and PSMF diet was calculated to be 60 and 25%, respectively, of the recommended dietary allowances (RDAs) for age and sex. Neither diet was supplemented with trace elements or calcium. Using a visual memory system, all subjects had a 24-h dietary intake recall before starting the weight-loss program. Iron, zinc, and copper intakes from the 24-h recall were compared with those from prescribed diets. Both diets produced a significant (p<0.001) weight reduction with a significant reduction in the arm muscle area of the PSMF group. After treatment, no significant change was observed in sFe, sFert, and TSI of either group, whereas eZn increased significantly in the HCBD and the PSMF groups (p=0.001 andp<0.006, respectively), with an improvement of the erythrocyte index (E.I.). A significant increase in pZn was also observed in the PSMF group (p=0.007). When compared with the usual intakes, HCBD supplied less iron (p=0.04) and more copper (p=0.001), whereas PSMF provided more zinc (p=0.026).     

Induction of 6-thioguanine-resistant mutants by hyperoxia and gamma-irradiation: effect of compromising cellular antioxidant systems

Hyperoxia and gamma-irradiation were found to be mutagenic in a transformed Syrian hamster cell line in a dose-dependent manner. The frequency of resistance to 6-thioguanine increased from 10 per 10(6) survivors after 48 h of growth in 70% O2 to 32.6 (highly significant) after 75 h. Increasing the oxygen tension to 95% resulted in a significant mutagenic response in only 44 h. At equitoxic doses, gamma-irradiation was 4 times more mutagenic than 70% O2. After growth in hyperoxia, the cells showed an enhancement of catalase activity, glutathione peroxidase activity and glutathione levels but there was little effect on superoxide dismutase activity. Diethyldithiocarbamate (3 mM, 1.5 h) was mutagenic in normoxia and potentiated the mutagenic activity of both gamma-irradiation and hyperoxia. Cells thus treated showed an 855 reduction in superoxide dismutase activity. When diethyldithiocarbamate was used in conjunction with a direct-acting alkylating agent, the mutagenic response was only additive. Depletion of cellular glutathione with buthionine sulfoximine (0.2 mM) or inhibition of catalase activity with aminotriazole (100 mM) was also effective in potentiating the mutagenic response of gamma-irradiation and hyperoxia. The data demonstrates that endogenously produced activated oxygen species are mutagenic to hamster cells in culture and suggest that aerobic organisms are subject to an unavoidable background risk due to living in an oxygen atmosphere.     

Mutagenicity studies on coffee. The influence of different factors on the mutagenic activity in the Salmonella/mammalian microsome assay

Recently, mutagenic activity on several strains of Salmonella typhimurium has been found in many heat-processed foodstuffs. The previously reported direct-acting mutagenic activity of coffee in Salmonella typhimurium TA100 (Ames assay) was confirmed in our study. In addition to TA100, a mutagenic effect of coffee was also found by using the newly developed strain TA102. The mutagenic activity was abolished by the addition of rat-liver homogenate. 10% S9 mix completely eliminated the mutagenic activity of 30 mg of coffee per plate. The addition of reduced glutathione to active S9 further decreased the mutagenic activity and also reduced the mutagenicity together with inactivated S9. The compound or compounds responsible for this inactivation are heat-labile and seem to be located in the cytosol fraction of the S9. Part of the mutagenicity of coffee was also lost spontaneously upon incubation at temperatures between 0 degrees and 50 degrees C. The loss of activity was dependent on temperature, being more pronounced at 50 degrees C compared to 0 degrees C (at 50 degrees C approximately 50% of the mutagenic activity was lost after 6 h). As anaerobic conditions prevented this loss of mutagenicity almost totally, oxidative processes are probably responsible for the inactivation. The stability of the mutagen was not influenced by incubation at low pH values (pH 1-3), with or without the addition of pepsinogen. The mutagenic properties of methylglyoxal, which to some extent could be responsible for the mutagenic activity of coffee, were compared with those of coffee. Methylglyoxal was strongly mutagenic towards Salmonella typhimurium TA100 and TA102. Its mutagenic activity was partially inactivated by the addition of 10% S9. Glyoxalase I and II together with reduced glutathione abolished the mutagenic activity of methylglyoxal but reduced the mutagenicity of coffee by only 80%. Since these enzymes occur in mammalian cells, the mutagenic compound(s) of coffee could also be degraded in vivo. This conclusion is supported by the fact that a long-term carcinogenicity study with rats was negative. These results clearly demonstrate that the effects observed in vitro do not necessarily also occur in vivo, but that in vitro experiments may contribute to the understanding of fundamental mechanisms of chemical carcinogenesis.     

Structural Requirements for Mutagenic Activities of N-Heterocyclic Bases in the Salmonella Test System

The mutagenic activities of quinoline, isoquinoline, phenanthridine, benzo(f)quinoline, benzo(h)quinoline and their α-amino derivatives were compared in relation to the effect of structural changes using the Salmonella typhimurium test system. All mutagenic compounds tested require the liver microsomal fraction for their mutagenic activity. Phenanthridine, two benzoquinolines and quinoline were mutagenic. α-Amination of two benzoquinolines and quinoline resulted to increase their mutagenic activity intensively. Addition of a benzene ring to the benzene moiety of 2-aminoquinoline, so that two carbon atoms are shared, affected distinctly the increase in the mutagenic activity. The co-existence of benzoquinoline series with 2-aminobenzo(f)quinoline showed the clear synergistic action.     

Mutagenicity in Salmonella typhimurium tester strains of XAD-2-ether extract, recovered from Katsura River water in Kyoto City, and its fractions

The mutagenic activity of XAD-2-ether extracts recovered from Katsura River water at monthly intervals during September to December 1980 was tested on S. typhimurium TA1538, TA1535, TA98 and TA100. The extracts showed strong mutagenic activity towards TA1538 nd TA98, especially in the presence of S9 mix. They were more active to TA1538 than to TA98. Some of each of the XAD-2-ether extracts were pooled and separated into neutral, basic and acidic fractions, and their mutagenic activities were tested on TA1538 and TA98 to determine their contribution to the total mutagenic activity of the parent extract. The neutral fraction was responsible for most of the total mutagenic activity of the parent extract. Although the basic fraction was only 5.4% by weight of the parent extract, it was much more mutagenic than any other fraction. Its contribution to the total mutagenic activity was higher than the acidic fraction which was 30.5% by weight of the parent extract.     

Dietary factors affecting the urinary mutagenicity assay system. I. Detection of mutagenic activity in human urine following a fried beef meal

Studies have been conducted to determine whether the mutagens in fried beef ingested by human subjects are excreted in the urine. Urine samples were collected from individuals on liquid or regular diets before and after a fried beef meal. The mutagenic activity of the samples was tested in the Ames Salmonella/microsome assay system. The results showed that in individuals on liquid diets, most of the urinary mutagenic activity is recovered within 2-6 h after consuming a fried beef meal. In one individual tested, mutagenic activity was found in urine samples obtained 6-15 h after the fried beef meal. No mutagenic activity was detected in any of the urine samples obtained 15-24 h following the meal. In individuals on a regular diet, however, mutagenic activity was frequently observed in urine samples obtained 16-24 h following the fried beef meal, although the mutagenic activity was not as great as that in the preceding 16 h. It appears that the mutagenic agents generated by the frying of beef are ingested, absorbed, and excreted by the human body in biologically detectable quantities. These results suggest that subjects should abstain from fried beef at least one day prior to and during urine mutagenicity screening.