Cytotoxicity of oxidants and asbestos fibers in cultured human mesothelial cells.

The authors investigated the mechanisms caused by oxidants (superoxide and hydrogen peroxide) and asbestos (amosite) fibers in human mesothelial cells. Immortalized human pleural mesothelial cells (MET 5A) were exposed in vitro to one of the following: hypoxanthine (100-200 microM) plus xanthine oxidase (10-20 mU/ml) as a superoxide-generating system, H2O2 (50 microM-5 mM); or amosite (1-100 micrograms/cm2). Cellular adenine nucleotide depletion, DNA single strand breaks, extracellular release of nucleotides, and their catabolites and lactate dehydrogenase (LDH) were assessed as markers of cell damage after 4-6 h exposure to the oxidants or fibers. The effect of intracellular antioxidant enzymes and exogenous antioxidants on cell damage were investigated during oxidant and amosite exposure. Superoxide radical and H2O2 exposure resulted in the depletion of adenine nucleotides, accumulation of the products of nucleotide catabolism, induction of DNA single strand breaks, and extracellular LDH release. Amosite exposure did not cause nucleotide depletion or induction of DNA single strand breaks. Inactivation of the intracellular antioxidant enzymes glutathione reductase or catalase augmented cell damage during H2O2 exposure but not during amosite exposure.

Mechanisms of DNA damage induced in rat hepatocytes by quinones.

Hydroquinone (HQ), catechol (CAT), duroquinone (DQ) and resorcinol (RES) have been shown to induce single-strand breaks (SSB) in DNA in isolated hepatocytes using the alkaline elution assay. The relative capacities of these agents to induce SSB were shown to be: DQ = HQ much greater than CAT greater than RES. Preincubation of hepatocytes with the Ca(2+)-chelator Quin-2 AM reduced the level of DNA damage to a great extent, as did treatment with DQ and RES, and, to a lesser extent, treatment with HQ and CAT. Preincubation with an inhibitor of poly(ADP-ribose) polymerase, 3-amino-benzamide, did not influence the extent of DNA damage caused by DQ and RES, but increased the damage caused by HQ and CAT. The conclusion of the present investigation was that the dominating reaction of HQ and CAT, with DNA is an arylation, whereas DQ and RES induce DNA damage by oxidative stress.

Single-strand breaks in DNA of various organs of mice induced by styrene and styrene oxide.

Styrene and its metabolite styrene oxide were given i.p. to mice. The induction of single-strand breaks (SSB) in DNA was studied with the DNA unwinding technique. The level of SSB in kidney-DNA was a linear function of the dose for both substances. Styrene and styrene oxide induced an increase in the level of SSB in DNA of kidney, liver, lung, testis and brain 1 h after administration. After 24 h the damage remained on an enhanced level in liver, lung and testis after styrene oxide administration and in all organs except liver after styrene administration.

Induction of single-strand breaks in DNA of mice after inhalation of vinyl chloride.

Female mice were exposed to 500 ppm vinyl chloride (VC) for 6 h/day 5 days/week for 1-8 weeks. Groups of mice were killed at different times during this period. DNA damage, expressed as single-strand breaks (SSB), was studied in liver, kidneys, lungs, spleen and brain. The level of SSB increased in liver, kidneys, spleen and lungs with time of exposure and reached a plateau for kidneys and lungs after 80 and 120 h of exposure. In spleen there was only a slight increase in the SSB, and in brain no detectable increase was found.

DNA damage induced by etoposide; a comparison of two different methods for determination of strand breaks in DNA.

Etoposide induces DNA damage to cells by interacting with the nuclear enzyme topoisomerase II. In this investigation the human lymphoblastic leukemia cell line (CEM) was used to study induction of DNA-strand breaks and cellular drug uptake after treatment with etoposide at a concentration of 0.5-2 micrograms/ml. High performance liquid chromatography was used for determination of etoposide concentrations. The alkaline elution assay and the DNA unwinding technique were compared for quantifying strand breaks in DNA induced by etoposide. The concentrations required to increase the level of DNA damage significantly was as follows: the DNA unwinding technique, 0.20 microgram/ml; the alkaline elution assay with proteinase K, 0.45 microgram/ml; the alkaline elution assay without proteinase K, 0.60 microgram/ml. When the half-life was adjusted, considering the efflux time of etoposide from cells, it was found to be only a few minutes. The present data show that the DNA unwinding technique is to be preferred for the screening of DNA damage. This technique is easier and quicker to perform than the alkaline elution technique.

Single-strand breaks, chromosome aberrations, sister-chromatid exchanges, and micronuclei in blood lymphocytes of workers exposed to styrene during the production of reinforced plastics.

Chromosome aberrations (CA), micronuclei (MN, cytokinesis-block [CB] method), and sister-chromatid exchanges (SCE) were analysed in blood lymphocytes of 17 workers and 17 control subjects. The mean urinary mandelic acid level (average 9.4 mmol/l) and styrene glycol in blood (average 2.5 mumol/l) implied exposure to about 300 mg/m3 of styrene in the plant. The number of CA was significantly higher in non-smoking workers compared with nonsmoking controls. A significant correlation was observed between duration of exposure and individual CA level of all workers. No significant effects were observed in MN or SCE. Single-strand breaks (SSB) in DNA of isolated lymphocytes were studied in nine of the workers and eight of the controls by the DNA-unwinding technique. The results showed an increase in SSB among the exposed workers. The present findings support earlier reports on the increase of structural CA in blood lymphocytes of workers in the reinforced plastic industry, and also show that SSBs are elevated in such workers.

Chemical reactivity and mutagenicity of some dihalomethanes.

Four dihalomethanes; dichloromethane, bromochloromethane, dibromomethane and diiodomethane, have been studied with respect to their reactivities towards nucleophilic compounds of different strengths in water solution and with respect to their toxicities and mutagenic effectiveness in bacterial test systems. The correlation between biological activity (toxicity and mutagenic effectiveness in Salmonella TA100) and reactivity towards strong nucleophiles indicates that reactions with nucleophilic groups of high reactivity in the biological material, possibly SH or amino groups in proteins, are involved in their mechanism of action.

Covalent binding of styrene and styrene-7,8-oxide to plasma proteins, hemoglobin and DNA in the mouse.

The extent of covalent binding to plasma proteins, hemoglobin and guanine-N-7 in DNA was determined after intraperitoneal administration of radiolabelled styrene and styrene-7,8-oxide to mice. The degree of alkylation increased non-linearly with the dose. It was proportionally higher after the highest doses of styrene-7,8-oxide while the reverse was observed with respect to the ability of styrene to alkylate plasma proteins and DNA. Thus, a dose dependence was indicated in the elimination of both styrene and styrene-7,8-oxide. A comparison of the degree of alkylation of plasma proteins, hemoglobin and guanine-N-7 in DNA suggests that the two compounds are about equally effective as alkylating agents in vivo at moderate dose levels. At high doses styrene-7,8-oxide is the more effective alkylator. The alkylation of DNA in liver, brain and lung after administration of styrene-7,8-oxide exceeded that in spleen and testis.

Induction of single-strand breaks in DNA of mice by trichloroethylene and tetrachloroethylene.

Trichloroethylene (TRI) (4-10 mmol/kg body wt) and tetrachloroethylene (PER) (4-8 mmol/kg body wt) were given to male mice by i.p. injection. The induction of single-strand breaks (SSB) in DNA of liver, kidney and lung was studied by the DNA unwinding technique. There was a linear increase of the level of SSB in kidney and liver DNA but not in lung DNA 1 h after administration. The damage was completely repaired 24 h after injection. The capability of TRI and PER to induce SSB in liver DNA is compared to that of three other substances, i.e., methyl methanesulfonate (MMS), styrene-7,8-oxide and styrene, which have been studied earlier by the same technique. The potency of the substances for induction of SSB was in the following order: MMS greater than styrene-7,8-oxide greater than styrene greater than PER greater than TRI.

Determination of reaction rate constants for alkylation of 4-(p-nitrobenzyl) pyridine by different alkylating agents.

The rate constants have been determined for the reaction between some different alkylating agents and 4-(p-nitrobenzyl) pyridine (NBP) in methanol. These constants have been compared with those for alkylation of aniline in water. All the constants were lower in methanol than in water but in different degrees. The rate constants of the different alkylating agents have been calculated at a nucleophilic strength n=2. The genetic risk defined as the degree of alkylation of a nucleophile (n=2) is equivalent to the rate constant kn=2 and the target dose. The dependence of the genetic risk on the rate constant (kn=2) is discussed.

Reaction of benzyl chloride with haemoglobin and DNA in various organs of mice.

[14C]Benzyl chloride was given i.v. to male mice and the elimination of the radioactivity from various organs was studied. The alkylation of guanine-N-7 of DNA in the same organs and the alkylation of haemoglobin were determined 1, 6 and 24 h after injection of benzyl chloride. There was a decrease with time of the degree of alkylation of DNA in brain, testis and liver, but an increase in lung and probably also spleen. The degree of alkylation of haemoglobin also increased. The expected degree of alkylation of DNA was calculated from the degree of alkylation of haemoglobin. The ratio of found and expected degree of alkylation of DNA was about 3 times higher for testis and brain than for the other organs 1 h after administration of benzyl chloride.

DNA damage in lung cells in vivo and in vitro by 1,3-butadiene and nitrogen dioxide and their photochemical reaction products.

A UV-irradiated mixture of 1,3-butadiene and nitrogen dioxide (NO2) was tested for its potency to induce DNA damage measured as single-strand breaks (SSB) in lungs of mice. Both gases were also tested separately. After 16 h exposure a UV-irradiated mixture of 40 ppm butadiene + 20 ppm NO2, but not 20 ppm butadiene + 10 ppm NO2 + UV, induced a significant increase in SSB as measured by the alkaline unwinding technique. There was no increase in the level of SSB using the alkaline elution technique during the same testing conditions. However, after 5 h exposure to 60 ppm butadiene + 30 ppm NO2 + UV both methods demonstrated a significant increase in SSB. Mice were also exposed to butadiene at 80 and 200 ppm for 16 h and at 500 ppm for 5 h. DNA damage was demonstrated in both liver and lung after 5 and 16 h (only at 200 ppm) of exposure using the unwinding technique. Using the alkaline elution assay, a significant increase in the level of SSB in lung and liver was found only after 5 h of exposure. When mice were exposed to 30 ppm NO2 for 16 h or 50 ppm for 5 h, a significant increase in SSB was found with the unwinding technique. Alveolar macrophages from mice were also exposed in vitro to the gas mixture and to butadiene and NO2 separately. In these experiments, the DNA damage was studied with the unwinding technique. A significant effect was demonstrated with 40 ppm butadiene + 20 ppm NO2 + UV. NO2 itself contributed to some extent to the increase. Reasons for the discrepancies between the unwinding and the alkaline elution techniques are discussed.

Biologic markers in ethylene oxide-exposed workers and controls.

Ethylene oxide (EtO) is an alkylating agent and a model direct-acting mutagen and carcinogen. This study has evaluated a panel of biologic markers including EtO-hemoglobin adducts (EtO-Hb), sister-chromatid exchanges (SCEs), micronuclei, chromosomal aberrations (CAs), DNA single-strand breaks (SSB) and an index of DNA repair (ratio of UDS to NA-AAF-DNA binding) in the peripheral blood cells of 34 workers at a sterilization unit of a large university hospital and 23 controls working in the university library. Comprehensive environmental histories were obtained on each subject including detailed occupational and smoking histories. Industrial hygiene data obtained prior to the study and personal monitoring during the 8 years preceding the study showed that workers were subject to low-level exposure near or below the current Occupational Safety and Health Administration (OSHA) standard of 1 ppm (TWA). Personal monitoring data obtained during 2 weeks prior to blood sampling were uniformly less than 0.3 ppm (TWA). After adjusting for smoking, EtO workplace exposure was significantly (p less than 0.001) associated with EtO-Hb (a carcinogen-protein adduct) and 2 measures of SCEs [the average number of SCEs/cell (SCE50) and the number of high frequency cells (SCEHFC)]. There was an apparent suppression of DNA repair capacity in EtO-exposed individuals as measured by the DNA repair index; i.e., the ratio of unscheduled DNA synthesis (UDS) and NA-AAF-DNA binding (p less than 0.01). No association of DNA repair index with smoking was found. Another important finding of this study is the highly significant correlation between EtO-Hb adduct levels and SCEHFC (p less than 0.01) and SCEs (p less than 0.02) which provides evidence of a direct link between a marker of biologically effective dose and markers of genotoxic response. In contrast, micronuclei, CAs and SSBs were not significantly elevated in the workers. The activity of the u-isoenzyme of glutathione-S-transferase (GT) was measured as a possible genetic marker of susceptibility and a modulator of biomarker formation. However, possibly because of confounding by age, no significant relationships were found between GT and any of the exposure-related markers by ANOVA or among other independent variables by regression. This study demonstrates significant effects of low-level EtO exposure, independent of smoking history, near or below 1 ppm on multiple biomarkers and suggests that the current OSHA standard may not be adequately protective. Previously described effects of smoking on EtO-Hb adducts, SCEs and SCEHFC were also seen in this study.(ABSTRACT TRUNCATED AT 400 WORDS)

Biomarkers in styrene-exposed boatbuilders.

14 fiberglass-reinforced plastics (FRP) boatbuilders were compared with 9 unexposed controls with respect to several chemical specific and nonspecific biomarkers measured in peripheral blood. Biomarkers included styrene-hemoglobin adducts (styrene-Hb), sister-chromatid exchanges (SCEs), micronuclei (MN), single-strand breaks (SSBs) and N-acetoxy-2-acetylaminofluorene-induced DNA binding (NA-AAF binding) as a measure of susceptibility to DNA damage. Workers' exposures averaged 11 ppm (8-h TWA; geometric mean) and ranged from 0.6 to 44 p.p.m. Mandelic acid levels were measured in end-of-shift urine samples and reflected an average styrene exposure equivalent to 15 p.p.m. There was a large though not significant difference in levels of styrene-Hb adducts among exposed workers and controls, largely the consequence of a single heavily-exposed individual with an extremely high level of adducts. Significant differences between biomarker levels in exposed workers and controls were observed with MN, SSBs and NA-AAF binding. No significant differences were seen in mean levels of SCEs nor in the incidence of cells with a high frequency of SCEs. The data suggest that exposure to levels of styrene in occupational settings near or below the current OSHA standard (50 p.p.m.) can induce damage at the cellular/molecular level. Appropriately-selected panels of biomarkers can be useful in identifying potentially harmful exposures.

Induction and time course of DNA single-strand breaks in lymphocytes from patients treated with dacarbazine.

Dacarbazine (DTIC) is an antitumor agent, used for the treatment of metastatic melanoma. It is metabolized to an alkylating agent which reacts with DNA. A fast and simple method was developed in order to measure drug-induced DNA damage in lymphocytes isolated from frozen blood samples of treated patients. The level of DNA damage was determined as single-strand breaks (SSB) by means of the alkaline elution technique using the fluorochrome Hoechst 33258. DTIC induced SSBs in lymphocytes. Most of the DNA damage was repaired after 20 h but after subsequent daily treatments there was an accumulation of SSB. The method described here can be used for monitoring DNA damage in lymphocytes of persons exposed to genotoxic compounds.

Single-strand breaks in DNA of various organs of mice induced by methyl methanesulfonate and dimethylsulfoxide determined by the alkaline unwinding technique.

The method for determination of single-strand breaks (SSB) in DNA by the technique of alkaline unwinding and hydroxylapatite chromatography has been applied for cell nuclei from organs of mice. Male mice were given methyl methane-sulfonate (MMS) and dimethylsulfoxide (DMSO) by i.p. administration. Cell nuclei were prepared from various organs and then lysed in alkali. The amount of DNA was determined by fluorometry using 4',6-diamidino-2-phenylindole.2HCl. The relative level of SSB in DNA was determined in various organs (liver, kidney, lung, spleen, testis and brain) 1-24 h after administration of the agent. After MMS-treatment the number of SSB in DNA increased to about the same extent in all organs 1 h post-treatment but then decreased by time. The SSB persisted for the longest time in brain- and lung-DNA. DMSO induced SSB only in DNA of kidney.

Exposure dependent increase in DNA single strand breaks in leucocytes from workers exposed to low concentrations of styrene.

Single strand breaks in DNA were monitored in leucocytes from 17 men occupationally exposed to styrene. Personal air monitoring was carried out during one workday with two diffusion samplers and a portable photoionisation detector placed in the breathing zone. Exposure to styrene was also monitored by analysing styrene in blood and urine and mandelic acid in urine. Single strand breaks were measured in leucocytes by the alkaline elution technique. The biological samples were collected before a shift, at the end of a shift, and the next morning, before the next shift. An exposure dependent increase in single strand breaks was seen at the end of a shift but not before a shift or the next morning. Linear regression analysis indicated that the amount of DNA damage was roughly doubled after eight hours of exposure to 18 ppm styrene or at a urine concentration of 240 mg mandelic acid/g creatinine compared with the damage in non-exposed men. This study indicates that monitoring of single strand breaks with the alkaline elution technique may be a sensitive marker of genotoxic effects. To our knowledge, this is the first time that such a marker has been shown to correlate with exposure to less than 20 ppm styrene.

Induction of single-strand breaks in liver DNA of mice after inhalation of vinyl chloride.

NMRI female mice were exposed to 100, 250 and 500 ppm vinyl chloride (VC). Cell nuclei were prepared from the liver, and single-strand breaks (SSB) were determined by the DNA unwinding technique. Haemoglobin (Hb) was isolated from the blood, and the degree of alkylation was determined as a measure of in-vivo dose by means of a gas chromatography-mass spectrometry (GC-MS) technique. A maximum level of SSB in liver DNA and of adduct levels of Hb was reached at 500 ppm, indicating that saturation of metabolic activation of VC had been achieved. The results demonstrate that VC induces SSB in liver DNA of mice in a dose-dependent manner and that about 80% of the damage is repaired within 20 h.

Single-strand breaks in DNA of peripheral lymphocytes of styrene-exposed workers.

Single-strand breaks (SSB) were determined by means of the DNA unwinding technique in peripheral lymphocytes of styrene-exposed workers and referents. A slight increase in SSB was observed among exposed subjects in comparison with a control group of office workers. A correlation was found between SSB, the excretion of mandelic acid and the concentration of styrene glycol in blood. The present work suggests that the DNA unwinding technique can be used for screening workers exposed to genotoxic compounds.