Metabolic activation of styrene by erythrocytes detected as increased sister chromatid exchanges in cultured human lymphocytes.

Styrene induces sister chromatid exchanges (SCEs) in human whole-blood lymphocyte cultures without exogenous metabolizing systems, which indicates that styrene is metabolically activated in this in vitro system. Whole-blood lymphocyte cultures from 11 male donors showed a clear increase in SCEs after a 48-hr treatment with styrene (2 mM) or with the reactive metabolite styrene 7,8-oxide (0.15 mM). Styrene (0.5 to 4 mM) induced a distinct dose-dependent increase of SCEs in whole-blood cultures (with 200 to 400 million red blood cells/ml) but only a slight effect in purified lymphocyte cultures (with 20,000 red blood cells/ml). SCE induction by styrene (2 mM) depended on the amount of red blood cells (0.02 to 2000 million/ml) added to the purified lymphocyte cultures. Cyclophosphamide, studied for comparison, clearly increased SCEs irrespective of the presence of erythrocytes. The results show that erythrocytes are essential for the activation of styrene in the lymphocyte test system. This activation probably results from the conversion of styrene into styrene 7,8-oxide by oxyhemoglobin.

Metabolic cytochrome P450 genotypes and assessment of individual susceptibility to lung cancer.

Three polymorphic cytochrome P450 genes that have attracted interest for their potential role in human pulmonary carcinogenesis, i.e. CYP1A1, CYP2D6 and CYP2E1, were studied in a population consisting of 106 lung cancer patients and 122 healthy controls. Polymorphism of the CYP2D6 gene encoding for debrisoquine hydroxylase was determined using XbaI restriction fragment length polymorphism (RFLP) analysis together with a PCR based method. All of the three most common presently known defective alleles of CYP2D6 were detected by this application. Subjects having genotypes either homozygous or heterozygous for the CYP2D6 wild type alleles were classified as extensive metabolizers (EMs) of debrisoquine whereas poor metabolizers (PMs) had two defective alleles. The PM individuals are thought to be less prone to develop lung cancer. The CYP1A1 and CYP2E1 genes were studied by RFLP analyses using Msp I and Dra I restriction enzymes, respectively, giving rise to two different sized hybridizable fragments in Southern blot analyses. In these RFPL analyses genotypes homozygous to the mutated allele have been presented as potent determinants of individual lung cancer risk. In the present study no association between polymorphic CYP1A1 and CYP2E1 genotypes and susceptibility to lung cancer was found. However, CYP2D6 polymorphism studies of the 122 healthy controls revealed seven poor metabolizer genotypes (5.7%), which compares well with the previously observed phenotypic distribution in the Finnish population, whereas only one PM genotype (1/106) was found among the lung cancer patients. These results agree with the previous suggestions that PMs of debrisoquine are less susceptible to lung cancer than EMs.

Chromosome aberrations and their relevance to metal carcinogenesis.

Scoring for structural chromosome abnormalities is one of the only practical methods available for detecting visual damage in human genetic material. Cytogenetic tests in vivo and in vitro have shown the clastogenic potential of a number of metals and metal compounds. The difficulties in in vivo studies lie in identifying a specific clastogen in an occupational setting, where simultaneous exposure to a number of organic and inorganic chemicals is a common phenomenon. Metals known to be carcinogens in animals also tend to possess chromosome-damaging properties, even though more extensive studies are needed before any conclusive evidence can be reached. The visible chromosomal damage produced by exposure to metal compounds should be considered as a warning indication of potentially adverse genetic and somatic effects in humans.

Exposure to PAH compounds among cokery workers in the oil shale industry.

The exposure of Estonian cokery workers to polynuclear aromatic hydrocarbons at an oil shale processing plant was assessed by occupational hygiene and biomonitoring measurements. To assess the external dose of exposure to polynuclear aromatic hydrocarbons, pyrene and benzo[a]pyrene concentrations were measured from the breathing zone of workers during a workshift. Skin contamination with pyrene and benzo[a]pyrene was assessed by skin wipe sampling. As a biomarker of exposure to polynuclear aromatic hydrocarbons and as an integral of all possible absorption routes of pyrene, 1-hydroxypyrene concentration was measured from post-shift urine samples. Eighteen percent of the personal air samples exceeded the Finnish threshold limit value of benzol[a]pyrene (10 micrograms/m3). Mean values for benzo[a]pyrene and pyrene were 5.7 micrograms/m3 and 8.1 micrograms/m3, respectively. Based on skin wipe sample analyses, the skin contamination was also obvious. The mean value of benzo[a]pyrene on the samples collected after the shift was 1.2 ng/cm2. In control samples, benzo[a]pyrene was not found. The mean value of urinary 1-hydroxypyrene concentration was 6.0 nmol/mmol creatinine for the exposed workers and 0.5 nmol/mmol creatinine for the controls. This study showed the usefulness of 1-hydroxypyrene as an indicator of internal dose of polynuclear aromatic hydrocarbons. We concluded that the cokery workers at the Kohtla-Järve plant are exposed to high concentrations of polynuclear aromatic compounds.

Inhalation exposure of rats and mice to 1,3-butadiene induces N6-adenine adducts of epoxybutene detected by 32P-postlabeling and HPLC.

In this paper we report DNA binding of butadiene monoepoxide, a first metabolite of 1,3-butadiene catalyzed by monooxygenases. We prepared alkylated purines as marker compounds for 32-P-postlabeling and electrochemical analysis and developed methods to measure the corresponding products. The traditional postlabeling assay was modified by incorporating a solid phase extraction column and high-performance liquid chromatography (HPLC) enrichment steps to the assay prior to labeling. The final analysis of adducted N6 adenines is based on two dimensional thin-layer chromatography (TLC) and an on-line HPLC/radioactivity analysis. The qualitative and quantitative results are based on positively identified marker compounds. Alkylated N7 guanines were released from DNA by neutral thermal hydrolysis, prepurified by HPLC, and analyzed by HPLC with a sensitive electrochemical detection procedure. By using these methods, we found alkylation of calf thymus DNA exposed to butadiene monoepoxide in vitro at adenine N6 and guanine N7 sites. Analysis of lung DNA samples from mice and rats exposed to butadiene through inhalation showed that adenine N6 adducts were formed in vivo in a dose responsive manner.

Micronucleus assay in lymphocytes as a tool to biomonitor human exposure to aneuploidogens and clastogens.

The analysis of micronuclei (MN) in cultured human lymphocytes is, in principle, able to detect exposure to clastogens and aneuploidogens alike. There is, however, no clear evidence from human biomonitoring studies or animal experiments showing that in vivo exposure of resting lymphocytes to an aneuploidogen could actually be expressed as MN in cultured lymphocytes. In vitro, a pulse treatment of human lymphocytes with vinblastine, an aneuploidogen, did result in MN induction even if performed before mitogen stimulation, although a much more pronounced effect was obtained in actively dividing lymphocyte cultures. On the other hand, it is probable that a considerable portion of "spontaneous" MN contain whole chromosomes, their contribution increasing with age. It also seems that cytochalasin B, used for the identification of second cell cycle interphase cells in the MN assay, is able to slightly increase the level of MN with whole chromosomes. If MN harboring chromosome fragments represent a minority of the total MN frequency, there may be difficulties in detecting a weak effect in this fraction of MN against the background of MN with whole chromosomes. This would reduce the sensitivity of the assay in detecting clastogens, unless MN with whole chromosomes and chromosome fragments are distinguished from each other. That a problem may exist in sensitivity is suggested by the difficulty in demonstrating MN induction by smoking, an exposure capable of inducing chromosome aberrations. The sensitivity of the lymphocyte MN assay could be increased by detecting kinetochore or centromere in MN, or by automation, allowing more cells to be analyzed.

Assessment of exposure to butadiene in the process industry.

Occupational exposure levels to 1,3-butadiene (BD) are variable but generally below 1 ppm in the European process industry. A preliminary analysis showed that hemoglobin adduct levels of butadiene monoxide (BMO) were increased among the worker groups with higher potential exposure to BD (process work, bomb voiding, repair duties) than among less exposed workers in maintenance and laboratory or control persons. In the same workers no exposure related effects were seen in the cytogenetic parameters studied, i.e. chromosomal aberrations, sister chromatid exchanges or micronuclei in peripheral blood lymphocytes. However, the glutathione-S-transferase polymorphism in the T1 gene might play a role in determining interindividual sensitivity to BD-induced chromosomal aberrations. Chromosomal aberrations (gaps excluded) were significantly (P < 0.05) increased among the workers lacking the GSTT1 gene as compared to the BD workers with the gene, while the other polymorphic GSTM1 gene showed no association with the cytogenetic parameters. More work needs to be done to study the adducts by other active BD metabolites than BMO and the role of the genetic polymorphisms controlling the variability of individual responses.

Effects of styrene oxide on chromosome aberrations, sister chromatid exchange and hepatic drug biotransformation in chinese hamsters in vivo.

In vivo inhalation exposure to styrene oxide (25, 50, 75 and 100 ppm) for 2, 4 or 20 days (25 ppm only) had no effects on chromosomal aberration rates or sister chromatid exchange (SCE) frequencies (BrdU/labelling performed in vitro) in the bone marrow cells of Chinese hamsters. The only positive response in aberration frequency was obtained when styrene oxide was injected in lethal concentration (500 mg/kg body weight, i.p.) into the animal. One animal out of six showed slightly elevated SCE values after this high dose. The response of the hepatic drug metabolizing enzymes to styrene oxide exposure was found to be rather weak, which may be due to rather high activity of epoxide hydratase in Chinese hamsters as compared to e.g. mouse.

Inability of styrene to induce nondisjunction in Drosophila or a positive micronucleus test in the Chinese hamster.

Styrene produced no effect on induction of sex-chromosome nondisjunction in Drosophila melanogaster fed on 500 ppm styrene for 24 h. No increase in the frequency of micronuclei from chromosome fragments or from nondisjunction of whole chromosomes was seen in the bone marrow of Chinese hamsters (Cricetulus griseus) injected (i.p.) with a single dose of 1.0 g/kg b.wt. styrene or with 250 mg/kg b.wt. of the primary metabolite, styrene oxide.

Chromosomal aberrations in bone marrow of Chinese hamsters exposed to styrene and ethanol.

No significant increase was detected in the number of chromosomally damaged cells in bone marrow of Chinese hamsters after either inhalation exposure to 300 ppm styrene or oral intake of 15% ethanol in drinking water (exposure time 4 days or 3 weeks). In combined exposure, a slight increase of chromosomal aberrations was observed in the group exposed for 4 days (2.0% aberrant cells vs. 0.0% in controls). The group exposed for 3 weeks to combined treatment showed no response (0.3% aberrant cells vs. 0.7% in controls).

Monitoring of occupational exposure to cytostatic anticancer agents.

Many anticancer agents have been shown to be carcinogenic, mutagenic and teratogenic in experimental animals and in in vitro test systems. Epidemiological data on the association of second neoplasms with a specific chemotherapy treatment is available on some 30 agents, and in the case of 10 compounds the overall evidence on human carcinogenicity has been evaluated to be conclusive (Group 1: IARC, 1987 and 1990). The primary source of human exposure to anticancer drugs is from their use in therapy of cancer. However, persons employed in the manufacture, preparation and administration of the drugs to patients and in nursing patients may also be exposed. Safe handling of anticancer drugs, since the introduction of various general handling guidelines, is now good practice in hospitals, pharmacies and drug manufacturing companies of most developed countries. Careless handling of cancer chemotherapeutic agents may lead to exposure of the personnel in amounts detectable with chemical or biological methods in the body fluids or cell samples of the subjects. The exposure is typically to mixed compounds over long-term and to low exposure levels with accidental peaks. Therefore, the use of biological exposure markers is appropriate for the monitoring of such exposure patterns. The biological markers/methods for exposure assessment are either non-specific (e.g., cytogenetic damage, point mutations or 32P-post-labelling adducts in peripheral blood lymphocytes, urinary mutagenicity) or specific for a given compound (immunological methods for DNA adducts, specific analytical methods). Studies have revealed minor amounts of cyclophosphamide in the urine of pharmacy technicians and nurses handling the drug even when taking special safety precautions (Sessink et al. (1994a) J. Occup. Med., 36, 79; Sessink et al. (1994b) Arch. Env. Health, 49, 165). Another study showed surface wipe samples with measurable cyclophosphamide even away from the handling site (McDevitt et al. (1993) J. Occup. Med., 5, 57). These studies strongly implicate the importance of skin absorption as an exposure route. Also accidental spillage is never completely avoidable (Sorsa et al. (1988) Mutation Res., 204, 465-479). The potential confounders (smoking etc.), toxicokinetics of the agent(s) to be assessed and individual working practices should be carefully considered in any exposure assessment studies using human body fluid samples. Environmental monitoring on indicator cytostatics should be combined into studies designed to identify potential occupational exposure situations to anticancer agents. A properly performed study should also include dissemination of information to the workers to create a psychologically positive atmosphere for this important work.

Interaction of Mesna (2-mercaptoethane sulfonate) with the mutagenicity of cyclophosphamide in vitro and in vivo.

The effects of sodium 2-mercaptoethane sulfonate (Mesna) on the mutagenicity of cyclophosphamide (CP) were assessed in vitro by the Ames test and in vivo in rats by analyzing micronuclei in bone marrow and mutagenic activity in urine. Mesna alone was negative in all test systems, while CP gave a positive response in all of them. In a combined treatment there was no significant reduction of the CP-induced mutagenicity in Salmonella. In rats the frequency of bone marrow micronuclei was not diminished when Mesna was given together with CP. May-Grunwald-Giemsa staining and Hoechst-Pyronin fluorescent staining techniques for micronuclei yielded similar results. The urine of rats treated with CP was mutagenic to Salmonella and no significant difference was observed when the rats had received both Mesna and CP. The results give support to the theory that Mesna acts primarily by reducing the toxicity of metabolites of CP, particularly acrolein, in the urinary tract and not by suppressing the mutagenicity of the active metabolites of CP.

Occupational exposure to anticancer drug--potential and real hazards.

Many anticancer agents have been shown to be mutagenic, teratogenic and carcinogenic in experimental systems and second malignancies are known to be associated with several specific therapeutic treatments. Anticancer agents thus represent a class of occupational carcinogens, the handling of which should involve no unnecessary exposure. The available methodologies to detect possible exposures from ambient air and from biological samples are discussed, and the published data on results are reviewed. Analytical methods are available for the detection of most frequently used anticancer drugs from all groups, i.e., alkylating agents, mitotic inhibitors, antimetabolites and antibiotics. The ambient samples taken from sites of admixture of cytostatics have often shown detectable, but low concentrations of anticancer agents. Urine samples from patients under chemotherapy as well as from personnel handling the drugs occupationally in hospitals have been analyzed both chemically and for excreted mutagenicity. Both cisplatin and cyclophosphamide have been detected in the urine of patients; furthermore, cyclophosphamide was observed in the urine of nurses who formulate and deliver this drug. Urinary mutagenicity assays have given both positive and negative results in various groups of nursing and pharmacy personnel. Cytogenetic methods have, likewise, been applied for monitoring purposes. Most of the available data concerns chromosome aberrations (CA) or sister-chromatid exchanges (SCE) induced in peripheral blood lymphocytes of patients under chemotherapy. A few studies on groups occupationally exposed to anticancer drugs have given positive results, but also negative reports have appeared for these same cytogenetic parameters. No studies are as yet available on the possible carcinogenic effects of occupational handling of anticancer drugs. Two recent case-referent studies among hospital personnel have pointed to slightly increased risks of disorders in pregnancy outcome; one of the studies has shown an excess of spontaneous abortions and other malformations in children of females with a history of work with anticancer agents.

1,3-Butadiene and its epoxides induce sister-chromatid exchanges in human lymphocytes in vitro.

Sister-chromatid exchanges (SCEs) were induced in human lymphocytes by 1,3-butadiene and its epoxides 3,4-epoxy-1-butene and 1,2:3,4-diepoxybutane. After a pulse treatment of 2 h, 1,3-butadiene produced a weak but reproducible increase in SCEs both with and without S9 mix. The response was similar in cultures of whole blood and of isolated lymphocytes. The 2 epoxide metabolites of butadiene, studied in whole-blood lymphocyte cultures without exogenous metabolic activation, were highly active SCE inducers. The lowest effective concentrations of butadiene, monoepoxybutene, and diepoxybutane were 2000 microM, 25 microM and 0.5 microM, respectively. A slight but dose-dependent increase in SCEs was also observed without an exogenous metabolic system after a 48-h treatment with 1,3-butadiene. Already the lowest concentration tested (500 microM) was effective. Again, the response was similar in cultures of whole blood and isolated lymphocytes, suggesting that the lymphocytes are capable of metabolically activating 1,3-butadiene.

Sister-chromatid exchanges induced by 1.3-butadiene and its epoxides in CHO cells.

Sister-chromatid exchanges (SCEs) were analyzed in CHO cells after pulse treatment with 1,3-butadiene, 3,4-epoxy-1-butene (monoepoxybutene) and 1,2:3,4-diepoxybutane (diepoxybutane). A weak dose effect was observed after exposure to 1,3-butadiene but only in the presence of S9 mix. Monoepoxybutene and diepoxybutane were highly effective in inducing SCEs at concentrations of 0.1-1 microM both in the presence and in the absence of S9 mix. At higher concentrations the response was more pronounced without S9 mix.

Application of a semi-automated SOS chromotest for measuring genotoxicities of complex environmental mixtures containing polycyclic aromatic hydrocarbons.

Soxhlet-extracted samples of standard reference materials (SRMs) 1649 (PAR1: urban dust/organics) and 1650 (PAR2: diesel particulate matter) from the U.S. Institute of Standards and Technology were tested for induction of SOS functions using a semi-automated version of the SOS chromotest with Escherichia coli PQ37. Concentrations of 10 polycyclic aromatic hydrocarbons in the extracts were determined using reversed-phase HPLC. Only the diesel particulate matter (PAR2) extracts expressed SOS induction activity, which decreased when metabolic activation was used. Mutagenic PAH compounds (e.g., chrysene) were found in higher concentrations in the PAR2 extracts than in the PAR1 extracts but this could not explain the genotoxicity while it was mainly exhibited without metabolic activation. The direct genotoxic activity of the diesel particulate matter sample PAR2 is probably caused by nitroaromatic compounds; this was also supported by parallel studies with the Ames/Salmonella assay.

Effect of emissions from residential wood stoves on SCE induction in CHO cells.

The SCE-induction capacity of emissions from an airtight horizontal baffled residential wood stove was investigated in CHO cells. The samples were taken under normal and starved air conditions, from burning birch and spruce separately. Both particle phase and vapour phase were collected. All samples induced a dose-related response in SCE both with and without a metabolic activation system, the rat-liver microsomal fraction. The burning conditions in the stove influenced the mutagenicity of the emissions more than the type of wood; the smoke from wood burning under starved air conditions was more than one order of magnitude more potent in inducing a significant SCE response. With all samples, the response in SCE induction was highest without metabolic activation. The toxicity of the samples, especially those without S9, limited the dose-range tested.

Mutagenicity in urine of workers in rubber industry.

Epidemiological studies have shown that those who work in rubber industry have an increased risk of cancer. In the working environment they are exposed, probably, to several hundred different chemicals some of them being known or suspected carcinogens and mutagens. The bacterial fluctuation test was used to detect the mutagenicity in the urines of exposed workers. A group of unexposed office clerks served as controls. Both groups consisted of smokers and non-smokers, and that was taken into consideration in the results. Rubber workers, either smokers or non-smokers, exhibited significantly higher mutagenic activity in their urine than the occupationally unexposed controls when the base-pair substitution strain E. coli WP2 uvrA was used as indicator organism. Use of the frameshift strain S. typhimurium TA98 revealed increased mutagenicity in the urine of occupationally exposed smokers, nonsmokers and unexposed smokers. The extent of mutagenicity in the urine of workers who smoked suggested a synergistic effect of smoking and occupational exposure. The bacterial fluctuation test with urine samples as sources of mutagenicity is able to detect chemical exposure if the excreted compounds are still in active form or can be activated. The method can be used to identify hazardous working conditions long before the manifestation of possible pathological changes in exposed individuals.

The parallelogram approach in studies of genotoxic effects.

Over the past two decades mutagenicity tests have been used for the identification of potential human mutagens and have had an ancillary role, as supportive evidence in the assessment of human carcinogens. The demonstration of human germinal mutagens has been beyond the main scope of short-term testing strategies. However, just as mutagenicity tests have been useful in detecting potential carcinogens so should carcinogenicity tests assist the identification of presumptive germ cell mutagens. Cancer is an easily observable phenotype of mutation for genotoxic carcinogens and multi-site carcinogens or gonadal carcinogens logically could be germ cell mutagens. Thus carcinogenicity and mutagenicity data for a given genotoxic chemical should be considered together in the identification of putative germinal mutagens. Clearly, most classified human carcinogens are genotoxic thus helping to build the case for human germ cell mutagenicity. This paper describes the issues involved in such thinking and suggests an enhanced parallelogram approach incorporating the cancer endpoint. The enhanced parallelogram is explored using 1,3-butadiene and ethylene oxide as examples. The obvious lack of data for extrapolations using the parallelogram method suggests the need for targeted studies specifically designed for use in this approach.

Induction of micronuclei and anaphase aberrations by cytochalasin B in human lymphocyte cultures.

The frequency of micronucleated cells in isolated 72-h human lymphocyte cultures treated with cytochalasin B (Cyt-B; 1.5-6 micrograms/ml for the last 28 h) was 9-21 times higher (mean 14.6 times) among multinucleate than binucleate cells. At 3 micrograms/ml, the concentration of Cyt-B originally recommended for the human lymphocyte micronucleus assay, the frequency of micronucleated multinucleate cells was 8.5%, while 0.7% of the binucleate cells had a micronucleus. Although no dose-dependent induction of micronuclei could be observed for either of the cell types, increase in the concentration of Cyt-B was associated with a decrease in the ratio of multinucleate to binucleate cells. Treatment with Cyt-B (1.5-12 micrograms/ml) increased the frequency of anaphase cells with aberrations, especially lagging chromatids. This finding was explained by a dose-dependent increase in multipolar (greater than or equal to 3 poles) divisions which had a high frequency of anaphase aberrations (39-53%), irrespective of the concentration of Cyt-B. Bipolar anaphases did not show a significant increase in aberrant cells, although a suggestive dependence on the concentration of Cyt-B was observed. The findings indicate that the high frequency of micronuclei in multinucleate lymphocytes produced by Cyt-B is due to mitotic errors arising when bi- (and multi-) nuclear cells divide. To avoid possible artifactually high micronucleus frequencies due to inclusion of cells that have divided greater than or equal to 2 times in the presence of Cyt-B, it is recommended that, in the human lymphocyte micronucleus assay using the cytokinesis-block method, the cell culture time is reduced to minimize the frequency of such cells and that only good preparations and regularly shaped binucleates are included in the analysis.