[Lesser validity of urinary benzene than S-phenylmercapturic acid for measuring occupational and environmental exposure to very low concentrations of benzene]. / Minore validità del benzene urinario rispetto all'acido S-fenilmercapturico nel rilevare l'esposizione occupazionale ed ambientale a concentrazioni molto basse di benzene.

AIM: To study the validity of urinary benzene as a biomarker of low and very low exposure to this toxicant, as compared with t,t-muconic acid (t,t-MA) and S-phenylmercapturic acid (SPMA), also taking into account the influence of cigarette smoking and co-exposure to toluene on the urinary excretion of benzene. MATERIALS AND METHODS: The results obtained in two different studies were compared: in the first, workers occupationally exposed to low concentrations of benzene (18 fuel tanker drivers and 23 filling station attendants) were compared with 31 controls and in the second, workers exposed to very low concentrations of benzene (the same 23 filling station attendants) were compared with the 31 controls. Exposure to airborne benzene and toluene was monitored with passive personal samplers (Radiello). Then the urine collected at the end of the work shift was analyzed for t,t-MA, SPMA and urinary benzene. All participants also filled out a questionnaire about their lifestyle habits. RESULTS: There were no differences among the three groups in terms of age and smoking habit. Occupational exposure to benzene and toluene and the urinary concentrations of t,t-MA, SPMA and urinary benzene were higher in the fuel tanker drivers than the filling station attendants and higher in the latter than in the controls. Cigarette smoking was found to be associated with urinary excretion of t,t-MA, SPMA and urinary benzene at both low and very low exposure to benzene. The biomarkers t,t-MA, SPMA and urinary benzene were almost always correlated, for both low and very low exposure to benzene. Notably, for low exposure to benzene a dependency relation was found with the levels of t,t-MA, SPMA and urinary benzene on both cigarette smoking and airborne benzene, whereas for very low exposure to benzene there was a dependency relation of SPMA on cigarette smoking and airborne benzene, of urinary benzene only on cigarette smoking and of t,t-MA on none of the variables considered. CONCLUSIONS: For occupational exposure to low concentrations of benzene, urinary benzene and SPMA showed a comparable validity, while for exposure to very low concentrations of this toxicant the validity of SPMA was confirmed while urinary benzene was found to be less useful. Cigarette smoking was the main factor conditioning the excretion of all the biomarkers of benzene in conditions of both low and very low exposure to the toxicant, so for the analysis of occupational exposure to benzene it is best to recommend abstention from smoking at least in the hours immediately before urine collection.

Applicability of urinary benzene to biological monitoring of occupational and environmental exposure to very low benzene concentrations.

OBJECTIVE: To verify whether urinary benzene is an applicable biomarker of occupational exposure to very low concentrations of benzene, considering the influence of cigarette smoke and benzene-toluene co-exposure. MATERIALS AND METHODS: 23 filling station attendants with occupational exposure to benzene and 31 controls were analyzed. Occupational and environmental exposure was monitored and t,t-muconic acid (t,t-MA), S-phenylmercapturic acid (SPMA), urinary benzene and creatinine in the urine samples were tested. RESULTS: Occupational exposure to benzene and toluene was significantly higher in the filling station attendants than in the controls, whereas t,t-MA, SPMA and urinary benzene were not different in the two groups. Instead, the smoker group showed significantly higher values for the above biomarkers than the non-smoker group, each of which included both exposed workers and controls. SPMA was dependent on airborne benzene and cigarette smoking, and urinary benzene only on cigarette smoking, while t,t-MA was not dependent on either of these variables. CONCLUSIONS: At very low concentrations of occupational exposure to benzene, urinary benzene is less valid than SPMA as a biomarker, even if both are strongly influenced by smoking habit. Abstention from smoking should therefore be recommended for at least two hours before urine collection.

In vivo CYP2E1 phenotyping as a new potential biomarker of occupational and experimental exposure to benzene.

Assessing CYP2E1 phenotype in vivo may be important to predict individual susceptibility to those chemicals, including benzene, which are metabolically activated by this isoenzyme. Chlorzoxazone (CHZ), a specific CYP2E1 substrate, is readily hydroxylated to 6-OH-chlorzoxazone (6-OH-CHZ) by liver CYP2E1 and the metabolic ratio 6-OH-CHZ/CHZ in serum (MR) is a specific and sensitive biomarker of CYP2E1 activity in vivo in humans. We used this MR as a potential biomarker of effect in benzene-treated rats and, also, in humans occupationally exposed to low levels of benzene. Male Sprague-Dawley rats (375-400g b.w.) were treated i.p. for 3 days with either a 0.5ml solution of benzene (5mmol/kg b.w.) in corn oil, or 0.5ml corn oil alone. Twenty-four hours after the last injection, a polyethylene glycol (PEG) solution of CHZ (20mg/kg b.w.) was injected i.p. in both treated and control animals. After 2, 5, 10, 15, 20, 30, 45, 60, 90, 120, 180, and 240min from injection, 0.2ml blood was taken from the tip tail and stored at -20 degrees C until analysis. A modified reverse phase HPLC method using a 5microm Ultrasphere C18 column equipped with a direct-connection ODS guard column, was used to measure CHZ and its metabolite 6-OH-CHZ in serum. No statistically significant difference in the MR was observed, at any sampling time, between benzene-treated and control rats. The concentration-versus-time area under the curve (AUC), however, was lower (p<0.05, Mann-Whitney test), whereas the systemic clearance was higher (p<0.05) in treated than in control rats. Eleven petrochemical workers occupationally exposed to low levels of airborne benzene (mean+/-SD, 25.0+/-24.4microg/m(3)) and 13 non-exposed controls from the same factory (mean+/-SD, 6.7+/-4.0microg/m(3)) signed an informed consent form and were administered 500mg CHZ p.o. Two hours later a venous blood sample was taken for CHZ and 6-OH-CHZ measurements. Despite exposed subjects showed significantly higher levels of t,t-MA and S-PMA, two biomarkers of exposure to benzene, than non-exposed workers, no difference in the MR mean values+/-SD was found between exposed (0.59+/-0.29) and non-exposed (0.57+/-0.23) subjects. So, benzene was found to modify CHZ disposition, but not CYP2E1 phenotype in benzene-treated rats, nor in workers exposed to benzene, probably due to the levels of exposure being too low.

Exposure to mutagenic airborne particulate in a rubber manufacturing plant.

Epidemiological studies conducted in the 1980s revealed that people working in the rubber manufacturing industry had an increased risk of cancer. Even now, workers employed in rubber processing are still at risk despite the measures adopted to improve their working conditions. The aim of the study was to evaluate the presence of a genotoxic risk in a rubber industry and to verify whether or not it was possible to locate the most dangerous position among the different rubber-working processes. The mutagenic activity of airborne particulate was evaluated in samples collected in the mixing department of a rubber manufacturing plant. Ambient air samples were taken over 3-h period in two stable positions near the mixing (Banbury mixer) and calendering areas. Personal air samples were taken over 2-h period during a normal workday from five workers employed in different rubber processing operations (mixing, weighing, calendering, compounding and extruding). The mutagenic activity of the air samples was determined by plate incorporation assay using Salmonella typhimurium strains (TA 98, TA 98NR, TA 100, YG 1021) with and without metabolic activation. Polycyclic aromatic hydrocarbon (PAH) concentrations were determined by high-performance liquid chromatography (HPLC); the presence of other presumable contaminants were carried out by gas chromatography-mass spectrometry (GC-MS). The results showed substantial direct and indirect frameshift mutagenicity in both ambient and personal samples. No mutagenic activity was present in S. typhimurium TA 100, except in the personal sample from a worker employed on the Banbury mixer. HPLC analysis revealed very low concentrations of PAHs. GC-MS analysis showed the presence of compounds such as azulene derivative, 1,2-dihydro-2,2,4-trimethylquinoline, N-methyl N-phenylbenzenamine, diphenylamine, bis(2-ethylhexyl)phthalate and bis(methyl-propyl)phthalate. We conclude that the high levels of mutagenic activity in ambiental and personal samples indicate the presence of substances with high genotoxic potency; no substantial differences were seen among the several rubber processing operations. PAHs were not involved in indoor pollution. GC-MS analysis revealed the presence of compounds which may be produced by high temperatures to which the raw materials are subjected during rubber manufacturing processes. These substances are potential carcinogen though their mutagen properties have not been clearly determined.

Urinary mutagenic activity after different immunosuppressive protocols in renal transplant patients.

Cyclosporin (CsA) and azathioprine (AZA) are useful immunosuppressive drugs in the management of kidney and liver transplant recipients. We investigated urinary mutagenicity in three groups of kidney transplant recipients after different immunosuppressive protocols. Urinary mutagenicity was detected in a base-pair strain, E. coli WP2uvrA, in a liquid incubation assay. No mutagenic activity was detected in the urines of patients treated with CsA (4.5 mg/kg); 85% of the urines in the second group treated with AZA (1.26 mg/kg) showed high mutagenic activity, whereas mutagenic activity was found in 40% of the urines of subjects treated with CsA and AZA (3.89 mg/kg + 1.15 mg/kg). These data suggest that immunosuppressive therapy with AZA carriers a high risk of urinary mutagenicity, while immunosuppressive combined treatment with CsA and AZA significantly reduces this risk.

Increases in polycyclic aromatic hydrocarbon content and mutagenicity in a cutting fluid as a consequence of its use.

The aim of this study was to evaluate the relationships between the length of time a cutting fluid was used, its content in polycyclic aromatic hydrocarbons (PAHs) and its mutagenic potential. The PAH concentrations were determined by means of a high-resolution gas chromatograph-mass spectrometer in samples of new cutting fluid and in samples used for 3, 6 and 9 months. The following PAHs were measured: phenanthrene, anthracene, fluoranthene, pyrene, benzo[a]anthracene, chrysene+triphenylene, benzo[e]pyrene, benzo[a]pyrene and perylene. Mutagenicity assays were carried out on the aforementioned samples using the Ames test. Salmonella typhimurium TA98 was used as an indicator to show up mutagens capable of inducing frame-shift genetic changes, and Escherichia coli WP2 uvrA was used as an indicator to detect mutagens capable of inducing base pair genetic changes. The mutagenic tests were carried out with and without microsomal activation, using 1:1, 1:10, 1:20 and 1:50 dilutions of cutting fluid samples. An increase in the concentrations of total PAHs over time was observed in the samples of cutting fluid used for 3, 6 and 9 months. The highest percentage increase in PAH concentrations was observed in the 6-month-old sample (10 times the initial concentration, from 45 to 411.8 micrograms of oil). None of the samples were mutagenic to S. typhimurium without metabolic activation or to E. coli with and without metabolic activation. All samples except for the 1:1 diluted sample showed moderate but significant mutagenic activity in the S. typhimurium test with metabolic activation.(ABSTRACT TRUNCATED AT 250 WORDS)

Mutagenic activity in wastewater concentrates from dye plants.

Wastewater concentrates from the wastewater treatment systems of three dye plants were tested for mutagenic activity in Salmonella typhimurium TA98 and Escherichia coli WP2uvrA using a fluctuation assay. Concentrates were prepared by passing samples of wastewater (5-6 or 30 litres) through two porous resins (XAD-2 and XAD-7) in series. S. typhimurium in the presence of microsomal activation proved to be the more sensitive marker of mutagenicity. Mutagenic responses were observed in concentrates from all three plants tested. The results show that mutagenic activity was particularly high in the incoming waters and increased after active, biological treatment. Physico-chemical treatment may be effective in decreasing mutagenic activity, but only if appropriately used.

Hepatic and intestinal effects of flurithromycin and erythromycin in the rat.

The effects of flurithromycin (30-100 mg/kg p.o.), a fluorinated macrolide, on rat hepatic enzymes and intestinal microflora have been compared with those of equal doses of erythromycin. This latter drug significantly decreases cytochrome b5, cytochrome P-450 and aminopyrine N-demethylase and moderately influences intestinal microbial flora. Flurithromycin showed almost opposite characteristics, with no hepatic interaction and marked effects on some bacterial species (e.g. Bacteroides) known to facilitate the colonization of pathogenetic bacteria.

Reference values of tests for the assessment of mutagenic activity.

The determination of mutagenic activity in biological media aims at detecting the exposure to mutagenic chemicals, or to chemicals transformed by the organism into mutagenic metabolites. Mutagenic activity is detected by various short-term tests which rely upon the interaction of the chemical with the DNA of, bacteria, Drosophila or mammalian cells. The urinary fluctuation test has been particularly useful in determining mutagenic activity in the urine of subjects exposed to low concentrations of suspected genotoxic chemicals. The assay procedure itself is relatively simple, the data, however, should be carefully evaluated in relation to the attributes of the donor, bearing in mind the confounding variables related to life-style, diet, occupation and drug intake.

Influence of glutathione administration on the disposition of free and total platinum in patients after administration of cisplatin.

The kinetics of platinum (Pt) was studied in 12 patients suffering from non-small-cell lung cancer or pleural mesothelioma. Each subject received an infusion of cisplatin (CDDP, 80 mg/m2), and six patients were pretreated with glutathione (GSH, 2.5 g given i.v.) at 15 min prior to the cisplatin infusion. After a 3- to 4-week interval, all patients were given a second course of treatment on the same schedule. A biexponential model was fitted to plasma concentrations of total and ultrafilterable Pt. The excretion of Pt in urine was evaluated during the first 48 h after the CDDP infusion. Following the administration of CDDP alone or with GSH pretreatment, the pharmacokinetic parameters of Pt did not significantly differ between the treatments. Also, the unbound fraction determined at each sampling time did not vary significantly between the treatments. However, it is noteworthy that the mean values obtained for the terminal half-life, the volume of distribution, the renal clearance, the percentage of the dose excreted in the urine, and the mean residence time of total Pt were higher in patients who had been pretreated with GSH, suggesting that GSH might increase both the rate of Pt elimination and the extent of Pt distribution and, as a consequence of the latter, might prolong the residence time of Pt in the body. In addition, the unbound fraction of Pt from the 4th to the 48th was higher following the first dose of CDDP+GSH than after treatment with CDDP alone. Because of the rather high variability in the values of the parameters obtained, further work is planned using a larger number of patients.

Indomethacin induced hepatic alterations in mono-oxygenase system and faecal Clostridium perfringens enterotoxin in the rat.

The administration of indomethacin to rats, at the dose of 10 mg/kg once daily for three days, caused a loss of microsomal cytochrome P-450 and cytochrome b5 in the liver, and a fall in drug-metabolizing enzyme activities (i.e. aminopyrine N-demethylase, NADP cyt. c. reductase). Indomethacin also induced intestinal lesions and a significant increase in Clostridium perfringens enterotoxin levels in the feces at 24 hours after both the second and third day of treatment. The above findings suggest that the development of intestinal lesion and the accompanying release of Clostridium perfringens enterotoxin, as well as hepatic enzyme alterations in the rat, result from indomethacin administration. Some of the data in this paper were presented at the Meeting of British Pharmacological Society in Ireland, July 6th-8th, 1988.

Urinary mutagenicity tests in lead-exposed workers.

Urinary mutagenic activity detected by the bacterial fluctuation assay, using Salmonella typhimurium TA98 and Escherichia coli WP2 uvrA with and without metabolic activation (S9 mix), was studied in a group of 21 workers exposed to inorganic lead and a control group of 22 non-occupationally exposed subjects. Occupational exposure to inorganic lead had no effect on urinary mutagenicity in the strains considered, with or without metabolic activation. In smokers (exposed and non-exposed), urinary mutagenic activity appeared to increase compared to non-smokers (exposed and non-exposed), only with Salmonella typhimurium TA98 in the presence of S9 mix.

Pharmacokinetics and human tissue penetration of flurithromycin.

The relationship between concentrations in serum and levels in tissue of flurithromycin, a new fluorinated macrolide, was determined in patients undergoing maxillofacial surgery and thoracotomy. All patients received 500 mg of flurithromycin orally every 8 h. Drug levels in serum, bone, soft tissue, lung, and pericardial fluid were determined microbiologically. The total amount of antibiotic per gram of tissue was calculated on the basis of the concentration in the supernatant of the homogenate. From the parallel course between free concentrations in serum and calculated contents in interstitial fluid tissue, it was concluded that the tissues examined were easily accessible by flurithromycin; penetration values measured by the ratio of areas under the curve were 8.3 for lung, 3.6 for bone, and 0.8 for soft tissue. The results of the pharmacokinetic study suggest that accumulation of the drug during repetitive multiple doses is predictable. Mean residence times were 10.2 and 8.3 h in groups 1 and 2, respectively. For bacteriostatic drugs such as macrolides, not only very high but also prolonged concentrations in tissue lead to favorable therapeutic result.