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.

The applicability of the measurement of urinary thioethers. A study of humans exposed to styrene during diet standardization.

The excretion of thioethers was measured in the urine of 6 volunteers, who were experimentally exposed to styrene, and 18 styrene workers. In addition, 12 clerks (non-smokers) and 12 sheet-metal workers (smokers) served as control groups. Diet was standardized during the experiments. Thioethers were measured by a spectrophotometric method. The volunteers were exposed to styrene, 210 mg/m3, for 2h at a 50-W workload. An increase in thioether excretion was observed; the largest was in the urine samples collected between 0.5 and 5 h after the end of the exposure. After 43 h the excretion of thioethers was close to the pre-exposure level (3.5 mmol/mol creatinine). About 1% of the styrene absorbed was detected as thioethers in urine, which is only about 1/10 of the conversion reported for rats. From excretion rate curves a half-life of about 11 h was calculated for styrene thioethers. The styrene workers were employed at two plants. The average exposure to styrene (time-weighted average 8 h) was estimated to be about 115 mg/m3 (smokers in plant A), 55 mg/m3 (non-smokers in plant A) and less than or equal to 10 mg/m3 (non-smokers in plant B). The excretion of thioethers in exposed workers at plant A was higher by 2-4 mmol/mol creatinine than that in non-exposed controls. In plant B, where exposure was lower, an increase in that amount of thioethers excreted in the urine by exposed workers was less pronounced, and was statistically significant only when post-shift samples were compared with pre-shift samples.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of specific mercapturic acids in human urine after experimental exposure to toluene or o-xylene.

Volunteers were exposed to toluene and o-xylene for 4 h at 300 mg/m3 and 350 mg/m3, respectively, in an exposure chamber. Urine voided during and after the exposure was analysed for S-benzyl-N-acetylcysteine (toluene mercapturic acid) and S-(o-methylbenzyl)-N-acetylcysteine (o-xylene mercapturic acid). The amount of S-(o-methylbenzyl)-N-acetylcysteine found in urine after exposure corresponded to a metabolic yield of less than about 0.01% of the total uptake. No S-benzyl-N-acetylcysteine was found. Thus, determination of specific mercapturic acids after exposure to toluene or o-xylene can hardly be used for genotoxic monitoring.

An improved standardized procedure for urine mutagenicity testing.

The aim of the present study was to optimize the procedures for urinary mutagenicity testing in order to lower the baseline variation in mutagenic activity found in urine from unexposed subjects and to increase the sensitivity of the method. This was accomplished by using urine from nonsmokers and smokers as well as chemically spiked nonsmokers' urine. Diet was standardized. The number of mutants per ml of urine calculated from the linear portion of the dose-response curve was used as a measure of mutagenicity. The parameters investigated were (i) the total volume of urine per resin volume, (ii) the flow rate, (iii) the pH, (iv) the ionic strength of the urine, and (v) elimination of histidine. XAD-2 and C18 Sep-Pak resins recovered mutagens in smokers' urine and in chemically spiked urine with the same efficiency when an optimized procedure was adopted. The optimized procedure using a maximum volume of 50 ml acidified urine per Sep-Pak cartridge, or equal amount of XAD-2 resin, gave well over ten times greater recovery of mutagens from smokers' urine than in earlier reports. Histidine was effectively eliminated, and the background variation was also lowered.

Influence of diet and other factors on urinary levels of thioethers.

Urinary excretion of thioethers has been used as an indicator of exposure to potentially alkylating agents in several studies. These studies, however, often had the disadvantage of high and varying background values. We have studied methods for sampling and determination of urinary thioethers. Modifications have been made making it possible to increase the sensitivity of the method substantially. The thioethers were extracted with ethyl acetate and hydrolyzed with sodium hydroxide. The thiols were then determined spectrophotometrically with the reagent of Ellman. The extraction procedure and the addition of the reagent were found to be critical steps, whereas the crude urine samples were comparatively stable during storage at -20 degrees C. Diet was found to be the most important factor. Using a standardized diet over 24 h with chicken, potatoes, bread and dairy products as the major components, excluding vegetables of the Cruciferae family, 24 subjects excreted low amounts of thioethers, 3.2 +/- 0.2 mmol/mol creatinine, (mean +/- SEM). Individual values did not exceed 6.5 mmol/mol creatinine. Without food restrictions the mean values and the standard deviations were increased up to five and ten times respectively. Ingestion of certain foodstuffs increased the thioether excretion up to 20 times. Twelve smokers (20 cig/d) excreted 6.7 +/- 0.4 mmol/mol creatinine (mean +/- SEM, afternoon samples and standardized diet). No effects of gender and age of the subject could be observed. It is recommended that diet factors be kept under strict control to increase the sensitivity of the thioether assay and to avoid misleading results due to diet effects.

Effects of exposure to vehicle exhaust on health.

Exposure to combustion engine exhaust and its effect on crews of roll-on roll-off ships and car ferries and on bus garage staff were studied. The peak concentrations recorded for some of the substances studied were as follows: total particulates (diesel only) 1.0 mg/m3, benzene (diesel) 0.3 mg/m3, formaldehyde (gasoline and diesel) 0.8 mg/m3, and nitrogen dioxide (diesel) 1.2 mg/m3. The highest observed concentration of benzo(a)pyrene was 30 ng/m3 from gasoline and diesel exhaust. In an experimental study volunteers were exposed to diesel exhaust diluted with air to achieve a nitrogen dioxide concentration of 3.8 mg/m3. Pulmonary function was affected during a workday of occupational exposure to engine emissions, but it normalized after a few days with no exposure. The impairment of pulmonary function was judged to have no appreciable, adverse, short-term impact on individual work capacity. In the experimental exposure study, no effect on pulmonary function was observed. Analyses of urinary mutagenicity and thioether excretion showed no sign of exposure to genotoxic compounds among the occupationally exposed workers or among the subjects in the experimental study.

Epidemiologic support for ethylene oxide as a cancer-causing agent.

The mortality and incidence of cancer in three groups of workers with occupational exposure to ethylene oxide have been assessed. Eight cases of leukemia have occurred among 733 ethylene oxide-exposed workers compared with an expected 0.8 cases. Six cases of stomach cancer have been reported compared with 0.65 cases expected. These epidemiologic results provide support for an increased risk of malignancy in individuals with extended and intermittent exposure to low concentrations of ethylene oxide.

15-Hydroxylation of 5 beta-cholestan-3 alpha-ol and 24 alpha-ethyl-5 beta-cholestan-3 alpha-ol in rat liver supernatants (18,000 X g).

4-14C-labeled-5 beta-cholestan-3 alpha-ol and 24 alpha-ethyl-5 beta-cholestan-3 alpha-ol were incubated with rat liver 18,000 X g supernatant fractions fortified with NADPH. Among the metabolites formed were the 15 alpha- and 15 beta-hydroxy derivatives of the two substrates. The identification of these metabolites with liquid chromatography, thin layer chromatography, and gas-liquid chromatography-mass spectrometry is described. The formation of 15 beta-hydroxylated metabolites exceeded that of 15 alpha-hydroxylated ones. The total yields of 15-hydroxylated compounds formed was of the order 0.5-1.0%. The 15-hydroxylated metabolites could not be detected after incubations with rat liver mitochondria or a soluble liver fraction or after incubations of 5 beta-cholestan-3 alpha-ol with soybean lipoxygenase and linoleic acid.

Side-chain oxidation of monooxygenated C27- and C29-steroids in rat liver mitochondria and 18000 x g supernatant.

The 24-, 25- and 26-hydroxylation of 4-cholesten-3 alpha-ol, 4-cholesten-3 beta-ol, 5-cholesten-3 alpha-ol, 5-cholesten-3 beta-ol, 5 alpha-cholestan-3 alpha-ol, 5 alpha-cholestan-3 beta-ol, 5 beta-cholestan-3 alpha-ol, 5 beta-cholestan-3 beta-ol, 4-cholesten-3-one, 5 alpha-cholestan-3-one, 5 beta-cholestan-3-one and the 24 alpha-ethyl derivatives of 5 alpha-cholestan-3 beta-ol, 5 beta-cholestan-3 alpha-ol, 5 beta-cholestan-3 beta-ol and 4-cholestan-3-one were studied in rat liver mitochondria (8500 x g sediment fractions fortified with isocitrate) and in rat liver microsomes (18000 x g supernatants supplemented with NADPH). In the mitochondria, all C27-substrates and probably all C29-substrates were found to be omega-hydroxylated. From 24 alpha-ethyl-5 alpha-cholestan-3 beta-ol and 24 alpha-ethyl-4-cholesten-3-one two omega-hydroxylated products were identified. All C27- but no C29-steroids were found to be 24- and 25-hydroxylated. The yield of omega-hydroxylated metabolites were much higher than those of the 24-, and 25-hydroxylated products. The omega-hydroxylation of the C29-steroids amounted to 3-50% of that found for the corresponding C27-steroids. In the 18000 x g supernatant only one substrate, 5 beta-cholestan-3 alpha-ol, was found to be 25- and 26-hydroxylated and no 24-hydroxylation of any steroid could be detected.

Oxidation of 3-oxygenated delta 4 and delta 5 - C27 steroids by soybean lipoxygenase and rat liver microsomes.

The formation of dioxygenated metabolites of cholesterol, epicholesterol (5-cholesten-3 alpha-ol) 4-cholesten-3 beta-ol, 4-cholesten-3 alpha-one and 4-stigmasten-3-one was studied after incubations with soybean lipoxygenase and linoleic acid. From cholesterol and epicholesterol were formed the 7 alpha-hydroxy, 7 beta-hydroxy-, 7 beta-hydroperoxy-, 7-oxo and 5,6-epoxy-derivatives as well as 6 beta-hydroxy-4-cholesten-3-one. All delta 4-steroids were hydroxylated in the 6 alpha- and 6 beta-positions. The ratios between the yields of 6 beta- and 6 alpha-hydroxylated metabolites varied between 3:1 and 2:1. Incubations with 4-cholesten-3 alpha-ol and 4-cholesten-3 beta-ol also afforded the 4,5-epoxides of these steroids. The ratios between the yields of the 4 beta, 5 beta- and 4 alpha, 5 alpha-epoxides were ca. 4:1 for 4-cholesten 3 beta-ol and ca. 3:2 for 4-cholesten-3 alpha-ol. With iron-supplemented microsomes from rat liver, the compounds formed were qualitatively and quantitatively the same as with soybean lipoxygenase, whereas with 18,000 X g rat liver supernatant fractions the yields of all products formed--except 7 alpha-hydroxycholesterol and 6 beta-hydroxy-4-cholesten-3-one--were markedly decreased. The results indicate the presence of a rat liver microsomal 6 beta-hydroxylase which can use 4-cholesten-3-one as a substrate and extend previous findings of similarities between soybean lipoxygenase and a nonspecific lipoxygenase in rat liver microsomes.

7-Hydroxylation of 3-oxygenated C27-, C28-, and C29-steroids in rat liver 18,000 g supernate.

Structurally closely related steroids have been tested as substrates for the NADPH-dependent cholesterol-and cholestanol-7 alpha-hydroxylase(s) considered to be the rate-limiting enzyme(s) in bile acid biosynthesis. Of the steroids tested, 5-cholesten-3 alpha-0l, 5 alpha-cholestan-3 alpha-ol, 5 beta-cholestan-3 alpha-ol, 5 beta-cholestan-3 beta-ol, 4-cholesten-3 alpha-ol, 4-cholesten-3 beta-ol, 5 alpha-cholestan-3-one, 5 beta-cholestan-3-one, 24 alpha-methylcholesterol and the 24 alpha-ethyl derivatives of cholestanol, 5 beta-cholestan-3 alpha-ol, 5 beta-cholestan-3 beta-ol, and 4-cholesten-3-one, only 4-cholesten-3 beta-ol was 7 alpha-hydroxylated to a significant extent (approximately 1/5 of the conversion of exogenous cholesterol). This suggests that the 7 alpha-hydroxylase(s) is sensitive to the structure of the side chain, and that it requires a rather flat steroid nucleus (delta4-, delta5-, or 5 alpha-steroid) and an equatorial or quasiequatorial hydroxyl group at C3. The nature of the 7 alpha-hydroxylation is discussed and the importance of the beta-side of the steroid molecule is emphasized. Minute amounts of the 7 beta-hydroxy derivatives were formed from 4-cholesten-3 beta-ol, 5 beta-cholestan-3 alpha-ol, 24 alpha-ethyl-5 beta-cholestan-3 alpha-ol and, probably, from 5 beta-cholestan-3 beta-ol and 24 alpha-ethyl-5 beta-cholestan-3 beta-ol.

Side chain hydroxylation of cholesterol, campesterol and beta-sitosterol in rat liver mitochondria.

The extent of the side chain hydroxylation of cholesterol, campesterol (24 alpha-methylcholesterol), and beta-sitosterol (24 alpha-ethylcholesterol) in rat liver mitochondria has been compared. Two beta-sitosterol metabolites, tentatively identified by liquid chromatography, thin-layer chromatography, gas-liquid chromatography, combined with radioactivity detection, and gas-liquid chromatography-mass spectrometry as the 26- and 29-hydroxy derivatives, were formed in the proportion 1:1. The sum of 26-hydroxy- and 29-hydroxy-beta-sitosterol obtained amounted only to about one-fourth of the yield of 26-hydroxycholesterol. Campersterol appeared to give rise only to 26-hydroxycampesterol (tentatively identified), which was formed in similar yields as 26-hydroxycholesterol (0.2-0.4%). The formation of 29-hydroxy-beta-sitosterol but not of 28-hydroxycampesterol indicates that the omega-hydroxylation of the steroid side chain is dependent on the length of the side chain. Cholesterol gave rise to identifiable amounts of a 25-hydroxy derivative but the formation of 25-hydroxy derivatives of beta-sitosterol and campesterol could not be established with certainty. 24-hydroxycholesterol was also found to be formed in the mitochondrial system. The ratio between the yields of 26- and 25-hydroxychoelsterol ranged between 2 and 3, and that between 26- and 24-hydroxycholesterol was about 10.

Conversion of 7alpha-hydroxycholesterol and 7alpha-hydroxy-beta-sitosterol to 3alpha, 7alpha-dihydroxy- and 3alpha, 7alpha, 12alpha-trihydroxy-5beta-steroids in vitro.

The metabolism of 7alpha-hydroxycholesterol and 7alpha-hydroxy-beta-sitosterol (24alpha-ethyl-5-cholestene-3beta,7alpha-diol) has been compared in rat liver subcellular fractions. 7alpha-Hydroxy-beta-sitosterol was shown to be metabolized in the same manner as 7alpha-hydroxycholesterol. Thus, the following C29 metabolites have been identified: 24alpha-ethyl-7alpha-hydroxy-4-cholesten-3-one, 24alpha-ethyl-7alpha,12alpha-dihydroxy-4-cholesten-3-one, 24alpha-ethyl-7alpha-hydroxy-5beta-cholestan-3-one, 24alpha-ethyl-5beta-cholestane-3alpha,7alpha-diol, 24alpha-ethyl-7alpha,12alpha-dihydrozy-5beta-cholestan-3-one, and 24alpha-ethyl-5beta-cholestane-3alha,7alpha,12alpha-triol. The C29 compounds were generally less efficient substrates. The most pronounced difference was noted for the delta4-3-oxosteroid 5beta-reductase. Thus, 7alpha-hydroxy-4-cholesten-3-one was three to four times as efficiently reduced as the C29 analog. The oxidation of the 3beta,7alpha-dihydroxy-delta5-steroid to the 7alpha-hydroxy-delta4-3-oxosteroid, the 12alpha-hydroxylation of the 7alpha-hydroxy-delta4-3-oxosteroid, and the reduction of the 7alpha-hydroxy-5beta-3-oxosteroid to the 3alpha,7alpha-dihydroxy-5beta-steroid occurred in up to two times better yields for the C27 steroids.

Formation and metabolism in vitro of 5,6-epoxides of cholesterol and beta-sitosterol.

The formation of 5alpha,6alpha- and 5beta,6beta-epoxides of cholesterol and beta-sitosterol in rat liver subcellular fractions has been studied. The results show that the epoxidation seems to occur only in connection with the nonspecific tissue oxidation of the sterols. The beta-epoxides were formed in three- to fourfold excess over the alpha-epoxides. Both cholesterol epoxides were efficiently converted by a microsomal hydrolase into the 3beta,5alpha,6beta-triol. The conversion was less extensive with beta-sitosterol epoxides, especially the beta-epoxide. The possible biological significance in the formation of the sterol epoxides and the triols was evaluated by their ability to inhibit the microsomal cholesterol 7alpha-hydroxylase. Only the cholesterol epoxides and especially the beta-epoxide were active in this respect.

Studies on the formation of C7-oxygenated cholesterol and beta-sitosterol metabolites in cell-free preparations of rat liver.

The microsomal fraction and the 18,000 g supernatant fluid obtained from livers from normal rats, cholestyraminetreated rats, or from rats with a bile fistula have been used to compare the 7alpha-hydroxylation of [4-(14)C]cholesterol and beta-[4-(14)C]sitosterol (24alpha-ethyl-cholesterol). It was not possible to increase the specific formation of 7alpha-hydroxy-beta-sitosterol above 0.05% with any of the preparations. This conversion was less than 1% of that found for cholesterol. The inhibitory effect of added 7-oxo- and 7beta-hydroxy-beta-sitosterol on the 7alpha-hydroxylation of cholesterol was found to be much less than that of the corresponding cholesterol compounds. 7alpha-Hydroxy-beta-sitosterol was without effect. It is concluded that the activity of the cholesterol 7alpha-hydroxylase is dependent upon the structure of the steroid side chain.