Attenuation of cytogenetic damage by 2-mercaptoethanesulfonate in cultured human lymphocytes exposed to cyclophosphamide and its reactive metabolites.

Cyclophosphamide (CP) is metabolized to the reactive intermediates, phosphoramide mustard (PAM) and acrolein (AC), which have generally different molecular binding targets. Sodium 2-mercaptoethanesulfonic acid (MESNA) has been used clinically to alleviate hemorrhagic cystitis caused by CP chemotherapy, has exhibited anticarcinogenic effects in rats exposed to CP during a long-term bioassay, and acts in the urogenital tract by reacting with 4'-OH-CP and AC. The purpose of this study was to: (a) compare the relative abilities of PAM and AC to induce cytogenetic damage and cytotoxicity in cultured human lymphocytes; (b) assess the efficacy of MESNA to attenuate the cytogenetic damage and cytotoxicity induced by CP, AC, PAM, and diethyl-4'-hydroperoxycyclophosphamide (DEHP-CP), an activated AC-generating compound; and (c) determine if concanavalin A-stimulated T-lymphocytes, which differentiate into suppressor cells upon lectin activation, exhibit any heightened cytogenetic sensitivity compared to a variety of cultured mammalian cells during exposure to PAM or AC as reported by other investigators. Purified mononuclear leukocytes were stimulated with concanavalin A and exposed to CP (0.5-2.0 mM) without an exogenous activation system, AC (0.001-40.0 microM), PAM (0.0014-27.1 microM), or DEHP-CP (0.1-100.0 microM) in the presence or absence of MESNA (1, 5, or 10 mM). All four compounds induced significant concentration-related increases in the SCE frequency, but only PAM was clastogenic. On an induced SCE/microM basis, PAM was about 130 and 193 times more potent than were DEHP-CP and AC, respectively. MESNA protected against the cytogenetic damage and cytotoxicity induced by the four compounds, but it was particularly effective against AC and DEHP-CP by abolishing SCE induction completely. SCEs and chromosome aberrations differed considerably in their induction kinetics in lymphocytes exposed to PAM, and these disparities suggested an uncoupling of the two phenomena. Although SCE induction was not consistently associated with cytotoxicity with the four agents, chromosome aberration induction coincided with an inhibition of cell cycle kinetics in PAM-treated cells. The exceptionally high SCE frequency of up to 21 times baseline in cells exposed to PAM indicates that T-suppressor lymphocytes stimulated with concanavalin A may be particularly sensitive to the DNA-damaging effects of PAM. Finally, these data suggest that the anticarcinogenicity of MESNA correlates with its ability to attenuate cytogenetic damage and cytotoxicity induced by reactive CP metabolites.

Effect of acrolein on phosphoramide mustard-induced sister chromatid exchanges in cultured human lymphocytes.

Although phosphoramide mustard (PM) is generally recognized as being the most genotoxic metabolite of cyclophosphamide (CP), the contribution of acrolein to the cytogenetic toxicity of CP is unclear. Besides covalently binding to DNA, acrolein can inactivate critical proteins necessary for replicative DNA synthesis, RNA transcription, cell membrane integrity, and metabolism of xenobiotic and endogenous substrates. Because enzymatic processes are involved in sister chromatid exchange (SCE) formation and DNA excision repair, we hypothesized that acrolein might modulate SCE induction by PM due to acrolein's high binding affinity for proteins and low molecular weight sulfhydryl compounds. Human mononuclear leukocytes were isolated on a Ficoll-Hypaque density gradient, and 10(6) cells were inoculated into 1.9 ml of complete medium. T-cells were stimulated to grow with 4 micrograms concanavalin A/ml, and 5-bromo-2'-deoxyuridine (5 microM) was added 24 h later. The cultures were then treated with PM (0.069 microM) in the absence or presence of diethyl-4'-hydroperoxy-CP (DEHP-CP), an activated acrolein-generating compound, at concentrations of 0.1, 1, or 10 microM for 48 h. Demecolcine (1.35 microM) was added for the final 4 h of culture. PM alone induced about a 2-fold increase in the SCE frequency (PM, 14.1 +/- 0.5 (SD) versus control, 7.7 +/- 0.4) without cell cycle inhibition or reduced mitotic activity. DEHP-CP induced a concentration-related increase in the SCE frequency of up to 1.6-fold without any significant cell cycle inhibition or lowered mitotic activity. When PM and DEHP-CP were combined, SCE induction was additive for all three DEHP-CP concentrations. Except at the highest molar ratio of DEHP-CP:PM (145:1), there was no evidence of cytotoxicity in the other treatment groups. These results suggest that acrolein has a diminished role in mediating the cytogenetic and cytotoxic effects of CP. In addition, enzymes associated with SCE formation and, by inference, DNA excision repair may not be particularly susceptible to acrolein-induced inactivation.

Sister chromatid exchange induction in human lymphocytes exposed to benzene and its metabolites in vitro.

Previous in vivo studies have shown that low-dose benzene exposure (10 to 28 ppm for 4 to 6 h) in mice can induce sister chromatid exchange (SCE) in peripheral blood B-lymphocytes and bone marrow as well as micronuclei in bone marrow polychromatic erythrocytes. Because benzene is metabolized to a variety of intermediate compounds and two of these, catechol and hydroquinone, have been reported to be potent SCE-inducers, it is possible that other known and proposed metabolites could have chromosome-damaging effects in lymphocytes. Induced SCE frequencies, mitotic indices, and cell cycle kinetics were quantitated in human peripheral blood T-lymphocytes exposed to benzene, phenol, catechol, 1,2,4-benzenetriol, hydroquinone, 1,4-benzoquinone, or trans,trans-muconic acid. Three proposed metabolites of phenol, 4,4'-biphenol, 4,4'-diphenoquinone, and 2,2'-biphenol, which can be generated by a phenol-horseradish peroxidase-hydrogen peroxide system were also examined. Benzene, phenol, catechol, 1,2,4-benzenetriol, hydroquinone, and 1,4-benzoquinone induced significant concentration-related increases in the SCE frequency, decreases in mitotic indices, and inhibition of cell cycle kinetics. Based on the slope of the linear regression curves for SCE induction, the relative potencies were as follows: catechol greater than 1,4-benzoquinone greater than hydroquinone greater than 1,2,4-benzenetriol greater than phenol greater than benzene. On an induced SCE per microM basis, catechol was approximately 221 times more active than benzene at the highest concentrations studied. trans,trans-Muconic acid had no significant effect on the cytogenetic parameters analyzed. 2,2'-Biphenol induced a significant increase in SCE only at the highest concentration analyzed, and 4,4'-biphenol caused a significant increase in SCE frequency that was not clearly concentration related. However, both 2,2'- and 4,4'-biphenol caused significant cell cycle delay and mitotic inhibition. 4,4'-Diphenoquinone caused only a significant decrease in mitotic activity. These data indicate that in addition to phenol, di- and trihydroxybenzene metabolites play important roles in SCE induction. Furthermore, the results suggest either that benzene alone can induce SCE or, a more likely possibility, that mononuclear leucocytes have a limited capability to activate benzene.

Sister chromatid exchange induction in mouse B- and T-lymphocytes exposed to cyclophosphamide in vitro and in vivo.

Cyclophosphamide (CPA) is known to exert greater toxic effects of B- than on T-lymphocytes in vivo. Both in vitro and in vivo CPA treatments were used to assess the possible cytogenetic basis for these observations. First, male C57BL/6 mouse lymphocytes were stimulated to divide in vitro with either phytohemagglutinin (T-cell mitogen) or lipopolysaccharide (B-cell mitogen), and were then treated with CPA (0.05 to 1.0 mM) and 5-bromo-2'-deoxyuridine (2 microM) at 24 hr. Cultures were harvested at 60 hr following a 4-hr treatment with demecolcine (1.35 microM). CPA caused concentration-related increases in sister chromatid exchange (SCE) up to 3 times control frequencies; the resulting SCE induction curves for B- and T-cells were sigmoidal and equivalent. Second, mice were given a single i.p. injection of CPA (0.5, 1.0, or 5.0 mg/kg). Blood was removed 24 hr later and cultured without additional CPA, as described above. Dose-related increases in SCE frequencies were seen for both T- and B-lymphocytes. CPA induced consistently 2.5 to 3.7 more SCEs in B-cells than in T-cells. Thus, B- and T-lymphocytes exhibited an equal sensitivity to CPA in vitro, but B-cells were more susceptible to the genotoxic effects in vivo.

Sister chromatid exchange induction in patients with anaplastic gliomas undergoing treatment with radiation plus diaziquone or 1,3-bis(2-chloroethyl)-1-nitrosourea.

Diaziquone (AZQ) (NSC 182986), a lipid-soluble benzoquinone derivative, is presently being tested in a Phase III clinical trial to determine its efficacy in patients with anaplastic gliomas compared to the more standard 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) treatment following whole-brain irradiation. These patients on single-drug chemotherapy allowed us to evaluate the effects of each agent on sister chromatid exchange (SCE) induction in vivo. Eight weeks following the final radiation treatment, patients were randomly assigned to one of two groups: (a) 200 mg BCNU/m2, i.v., every 8 weeks; of (b) 15 mg AZQ/m2/day, i.v., for 3 consecutive days, every 4 weeks. Blood (5-10 ml) was drawn by venipuncture before treatment, within 10 h after treatment, and for two BCNU-treated patients at various other times. Peripheral blood lymphocytes were cultured by standard techniques for analysis of SCE. Eight weeks after irradiation but before chemotherapy, the mean SCE frequency in the patients' peripheral blood lymphocytes was 9.6 SCEs/metaphase. Following treatment with AZQ or BCNU, the baseline SCE frequency was increased more than 2-fold or 3-fold, respectively. Two months after BCNU treatment, there was less than a 25% reduction in SCE levels compared to samples taken and cultured within 10 h after treatment. These data show that lesions leading to SCE in human peripheral blood lymphocytes are relatively longlived, and that on a mg/m2 basis, AZQ is a more potent inducer of SCE in vivo than is BCNU.

Cytokine induction in human epidermal keratinocytes exposed to contact irritants and its relation to chemical-induced inflammation in mouse skin.

In response to exogenous stimuli such as phorbol-12-myristate 13-acetate, ultraviolet B radiation, and lipopolysaccharide, human keratinocytes produce soluble mediators that are important in primary contact irritancy including cytokines that are associated with proinflammatory properties (interleukin-1 alpha [IL-1 alpha], tumor necrosis factor alpha), chemotaxis (IL-8), and growth activation (granulocyte/macrophage colony stimulating factor, IL-6, transforming growth factor alpha). We examined qualitative and quantitative changes in selected intracellular and secreted cytokines in human keratinocyte cultures in response to non-sensitizing contact irritants (croton oil, sodium lauryl sulfate, methyl salicylate, ethyl phenylpropiolate), sensitizing irritants (oxazolone, dinitrofluorobenzene), and ulcerative agents (phenol, benzalkonium chloride, chromium trioxide). The chemicals were also applied to mouse skin to assess whether the chemical-specific pattern of inflammation correlated with the in vitro production of keratinocyte-derived cytokines. Although all agents elicited neutrophils to the site of chemical application, time dependent and chemical-specific patterns of inflammation could be detected. Sodium lauryl sulfate, phenol, and croton oil induced increases in IL-8 production at non-cytotoxic concentrations in semi-confluent human keratinocyte cultures. Phenol and croton oil stimulated tumor necrosis factor alpha production, whereas croton oil was the only agent found to induce granulocyte/macrophage colony-stimulating factor production. Croton oil, phenol, benzalkonium chloride, and dinitrofluorobenzene induced the intracellular production of IL-1 alpha without a concomitant release into the medium. The release of cytokines occurred in parallel with a relative increase in cytokine-specific mRNA transcripts. Studies using neutralizing antibodies to tumor necrosis factor alpha and IL-1 alpha demonstrated that IL-8 induction by croton oil and phenol occurred directly rather than through autocrine circuits. These data suggest that a given pattern of cytokine production is chemical-specific and may predict the contribution of keratinocytes to skin inflammation.

Analysis of cytogenetic damage in rat lymphocytes following in vivo exposure to nitrobenzene.

Male F-344 rats were exposed to target concentrations of 0, 5, 16, or 50 ppm nitrobenzene for 6 h/day, 5 days a week, for 21 days during a 29-day period. Isolated spleen (ISLs) and peripheral blood lymphocytes (PBLs) were cultured in the presence of 2 microM 5-bromodeoxyuridine and scored for sister-chromatid exchange (SCE) and PBLs were also scored for chromosome aberrations. No significant increase in SCE frequency or chromosome aberrations was found in the PBLs, and no significant increase in SCEs was observed in the ISLs at any of the concentrations. Thus, cytogenetic analysis of ISLs and PBLs provide no evidence of a genotoxic potential for nitrobenzene.

Role of keratinocyte-derived cytokines in chemical toxicity.

Following appropriate stimulation, such as with tumor promoters, ultraviolet light or various chemical agents, keratinocytes synthesize and secrete cytokines which can mediate or participate in dermatotoxic responses such as inflammation, hyperkeratosis, hypersensitivity and skin cancer. We have determined the qualitative and quantitative cytokine response in primary human keratinocyte cultures following exposure to several non-sensitizing contact irritants, sensitizers and ulcerative agents as well as a skin carcinogen. The chemicals were also administered to mice to assess whether the dermatotoxic response correlated with the in vitro production of keratinocyte-derived cytokines. Due to the complex cellular interactions that occur in the skin, it was not possible to identify specific cytokine profiles for most of the classes of dermatotoxic agents studied. However, the non-sensitizing contact irritants produced relative increases in the synthesis and secretion of the proinflammatory cytokines, interleukin-1 and tumor necrosis factor-alpha, as well as the neutrophil chemotactic cytokine, interleukin-8 compared to the other chemical agents. While ulcerative compounds as well as irritants elicited neutrophils to the site of chemical application when applied to the mouse skin, time-dependent and chemical-specific patterns of inflammation were detected. Treatment of human keratinocyte cultures with arsenic, a human skin carcinogen, resulted in a unique cytokine profile characterized by induction of growth factors, including transforming growth factor-alpha and granulocyte-macrophage colony stimulating factor. Treatment of v-Ha-ras transgenic mice, an animal model for skin cancer, with arsenic caused an increase in the number of papillomas as well as overexpression of these growth factors suggesting that they participate in arsenic-induced skin papilloma development. These studies indicate a diverse role exists for keratinocyte-derived cytokines in dermatotoxic actions.

A cytogenetic approach for detecting the selective toxicity of drugs in avian embryonic B and T lymphocytes.

The developing immune system of late stage embryos and neonates may be particularly susceptible to the toxicity of drugs and environmental contaminants due to high rates of cell proliferation and ongoing processes of differentiation. We have developed a cytogenetic assay to study the mechanisms of the selective targeting of cyclophosphamide (CP) to B lymphocytes compared to T lymphocytes in chicken embryos at days 18-19 of incubation. 5-Bromo-2'-deoxyuridine (BrdU; 3 mg/200 microliters PBS; two doses; 3-h interval) was pipetted onto the inner shell membrane in order to label DNA of replicating lymphoid cells. CP (1.25-40 mg/kg) was injected 1 h after the initial BrdU dose, and the embryos were exposed to colcemid (10 micrograms/100 microliters H2O) at hour 17. Three hours later, the bursa and thymus were removed, and the lymphocytes were swollen in hypotonic solution, fixed, and processed through a fluorescence-plus-Giemsa technique to differentiate sister chromatids. Based on reductions in mitotic indices, B cells were approximately 213 times more susceptible than T cells to the cytotoxicity of CP. Because the mitotic indices of B and T cells were comparable (21.3 +/- 3.7%, vs. 25.5 +/- 6.9%), the differential toxicity cannot be ascribed to greater numbers of B cells being in mitosis. CP induced a dose-related increase in the sister-chromatid exchange frequency in B cells of up to 10.4-fold above controls, representing one of the most sensitive vertebrate systems for detecting the genotoxicity of CP. The average generation time was slowed from 9.8 +/- 0.3 h in control B cells to 19.4 +/- 0.9 h in embryos exposed to 10 mg CP/kg. Furthermore, an analysis of control SCE data from 56 embryos indicated that there was a significant overdispersion of B cells exhibiting relatively high SCE frequencies compared to a Poisson distribution. Our data indicate that the chicken embryo in the late developmental stage is a good model for detecting the presence and selective toxicity of drugs and environmental toxins in differentiating B and T lymphocytes in vivo.

The effect of washing lymphocytes after in vivo treatment with streptonigrin on the yield of chromosome and chromatid aberrations in blood cultures.

Cytogenetic analyses of cultured blood lymphocytes from streptonigrin-treated female rabbits demonstrated that the yield of chemically-induced chromatid aberrations, expecially exchanges, is influenced by the method of blood handling before in vitro culture. When blood cells from treated animals were washed with tissue-culture medium before culture, chromatid breaks were observed at the same level as in the controls, and no chromatid exchanges were found. However, when duplicate aliquots of blood were inoculated directly into culture medium, without washing, elevated levels of chromatid breaks were observed and chromatid exchanges were found. These findings indicate that the clastogen, streptonigrin, was carried into the culture medium with the blood-cell inoculum.

Cytogenetic mechanisms in the selective toxicity of cyclophosphamide analogs and metabolites towards avian embryonic B lymphocytes in vivo.

Cyclophosphamide (CP) is selectively toxic to avian and mammalian B lymphocytes, but the mechanisms of action are incompletely understood. We used a structure-activity approach to determine the cytogenetic mechanisms underlying the selective lymphoid toxicity in chicken embryos at 18-19 days of incubation. Two doses of 5-bromo-2'-deoxyuridine (BrdU; 3 mg/200 microliters x 2) were pipetted onto the inner shell membrane to label lymphocyte DNA over 20 h. A single dose of the CP analogs or metabolites was given 1 h after the initial BrdU application. After a terminal 3-h exposure to demecolcine to block cells in metaphase, the embryos were sacrificed at hour 20, and their bursae and thymi were removed for cytogenetic processing. Microscope slide preparations of metaphases were stained by the fluorescence-plus-Giemsa technique to differentiate the sister chromatids for an assessment of sister-chromatid exchange (SCE) induction and cell cycle progression based on replication cycle-specific staining patterns. Isophosphamide (1.25-40 mg/kg), phosphoramide mustard (0.7-45.7 mg/kg), and 4-methylcyclophosphamide (1.3-42.1 mg/kg) selectively damaged B cells as shown by dose-related reductions in the mitotic activity, inhibition of cell cycle kinetics, and approximately 9-15-fold increases in the SCE frequency above control. B cells were up to 392 times more susceptible to the toxicity of these three bifunctional alkylating agents compared to T cells based on reductions in the mitotic activity. At most of the drug doses tested, the T-cell mitotic index was not depressed significantly and was usually higher than the control value by as much as 50-60%. Importantly, monochloroethylcyclophosphamide (70-245 mg/kg; monofunctional alkylation) did not induce differential lymphoid toxicity, although a 9-fold increase in the SCE frequency of B cells was observed at the highest dose. Didechlorocyclophosphamide (181-422 mg/kg; acrolein generation only) was a weak SCE inducer (approximately 1.8-fold increase) and was not selectively toxic to B cells. Our data show that selective toxicity to B lymphocytes is strongly associated with bifunctional alkylation via the chloroethyl groups rather than with monofunctional alkylation and acrolein-mediated damage. In addition, the results with phosphoramide mustard and 4-methylcyclophosphamide emphasize that aldehyde dehydrogenase activity is not the primary determinant in the relative sparing of T lymphocytes in vivo.

Induction of sister-chromatid exchange (SCE) and cell-cycle inhibition in mouse peripheral blood B lymphocytes exposed to mutagenic carcinogens in vivo.

To determine the sensitivity of the mouse peripheral blood lymphocyte (PBL) culture system, male B6C3f1 mice were injected i.p. with either 2-acetylaminofluorene (AAF) (20, 40, 80, 160 mg/kg), benzo[a]pyrene (BP) 25, 75, 150, 300 mg/kg), dichlorvos (DCV) (5, 15, 25, 35 mg/kg), ethyl methanesulfonate (EMS) (10, 30, 90, 180, 270 mg/kg), or N-nitrosomorpholine (NM) (37.5, 75, 150, 300 mg/kg) dissolved in either RPMI 1640 (DCV, EMS, NM) or sunflower oil (AAF, BP). 24 h later blood was removed by cardiac puncture, and the lymphocytes were cultured in the presence of lipopolysaccharide for analysis of SCE in B lymphocytes. All 4 mutagenic carcinogens (AAF, BP, EMS, NM) induced significant dose-related increases in SCE frequency. DCV, a potent neurotoxicant, caused no change in the baseline SCE frequency. At the highest concentration of each chemical examined, AAF caused a 1.6-fold increase, EMS a 1.8-fold increase, NM a 3.0-fold increase, and BP a 3.1-fold increase in SCE frequency compared to concurrent controls. A comparison of these results for PBLs with those reported in the literature for bone marrow cells indicates that PBLs offer a good quantitative and qualitative estimate of the SCE-inducing potential for these 5 compounds in bone marrow cells.

Implications of an elevated sister-chromatid exchange frequency in rat lymphocytes cultured in the absence of erythrocytes.

One important variable in complex culture systems such as whole blood is the interaction of the cell types present. To investigate the effects of erythrocytes (RBCs) and monocytes on the sister-chromatid exchange (SCE) frequency, Ficoll-Hypaque-separated Fischer-344 rat leukocytes were added to 1.9 ml of culture medium containing either 4 micrograms phytohemagglutinin or 4-8 micrograms concanavalin A/ml. Bromodeoxyuridine (BrdU;2 microM) was added at 24 h, and the cultures were harvested at 54 or 72 h. SCE frequencies in the mononuclear leukocyte cultures were consistently about 1.5- to 2-fold higher than in the whole-blood cultures. The titration of rat or human RBCs (0.05-2.5 X 10(9)) into purified rat leukocyte cultures reduced the SCE frequency to that of whole-blood cultures. Monocyte depletion decreased the elevated SCE frequency by approximately 50%. Scintillation counting of [14C]BrdU uptake in isolated RBCs revealed that less than 8% of the total amount of BrdU was sequestered. Also, BrdU induced a concentration-dependent increase in SCE in purified leukocytes, but the absolute increase was no greater than in whole-blood lymphocytes. Thus, BrdU had a minor role in the elevated SCE frequency in purified lymphocytes. Neither anti-oxidant enzymes such as catalase and superoxide dismutase nor the hydroxyl radical scavenger, dimethyl sulfoxide, decreased the SCE frequency. Although purified human lymphocytes had a small, but significant increase in SCE compared to whole blood, the magnitude of the dichotomous response between man and rat may represent a fundamental species difference.

Evaluation of chemically induced cytogenetic lesions in rabbit oocytes. I. The test system and the effects of streptonigrin.

An appropriate method for evaluating transmissible genetic damage in female germ cells is presented. Analysis of karyotype preparations from preimplantation (6- day) rabbit embryos for consistent structural chromosome abnormalities is described as a sensitive way in which these determinations can be made. A table prepared from summarized research reports shows that less than 1 in 2000 rabbit blastocysts karyotyped thus far has a consistent structural chromosome abnormality. In the new data presented in this study, none of the 90 control blastocysts karyotyped had consistent structural chromosome abnormalities while 13 of 278 from female rabbits treated with streptonigrin (NSC-45383) before conception did. These findings demonstrate that karyotype analysis for consistent chromosome lesions in preimplantation rabbit embryos from females exposed to chemical clastogens can serve as a sensitive and quantitative means of estimating effects on oocytes. The results of the study also show that the consistent structural chromosome lesions in the blastocyst karyotypes are what would be predicted from the mode of action of streptonigrin and the segregation of chromosomes during meiosis. The relative efficiency of this system based on the rabbit as compared to another recently described system bases on the hamster is also discussed.

Analyses of sister-chromatid exchange and cell-cycle kinetics in mouse T- and B-lymphocytes from peripheral blood cultures.

A reliable mouse peripheral blood lymphocyte culture assay has been developed for sister-chromatid exchange analysis. Crucial aspects for optimal mitogenesis include: (1) the addition of 5 X 10(5) leucocytes/ml culture; (2) the use of animals with leucocyte counts from 5 to 7 X 10(6)/ml; and (3) the addition of 6% mouse plasma for the first 24 h of a total 54-h incubation. When 7 micrograms phytohemagglutinin/ml were used to stimulate T-lymphocytes, the mitotic index was 3.4 +/- 0.3%, 28 +/- 2.3% of the metaphases were in first-division, and the SCE frequency/metaphase was 7.3 +/- 0.2 (n = 14 mice). When B-lymphocytes were stimulated with 60 micrograms lipopolysaccharide/ml, the mitotic index was 4.5 +/- 0.3%, 64 +/- 3.3% of the metaphases were in first-division, and the SCE frequency/metaphase was 4.6 +/- 0.1 (n = 7 mice). This culture method consistently yields sufficient numbers of metaphases from both B- and T-lymphocytes for SCE and chromosome-breakage studies.

Assessment of the genotoxic effects of methyl chloride in human lymphoblasts.

The activity of methyl chloride was measured in 4 genotoxicity assays. In an established human lymphoblast line, a 3-h treatment with 0-5% methyl chloride resulted in a dose-related increase in mutant fraction at the thymidine kinase locus and induction of sister-chromatid exchange. No increase in DNA damage, as measured by alkaline elution, was detected in the lymphoblasts at concentrations of methyl chloride shown to be mutagenic. Also, a concentration-related increase in 8-azaguanine-resistant fraction in Salmonella typhimurium was observed following a 3-h treatment with atmospheres containing 0-20% methyl chloride. Thus, methyl chloride is a weak, direct-acting mutagen for bacteria and human cells in culture.

Sister-chromatid exchange induction in peripheral blood lymphocytes of rats exposed to ethylene oxide by inhalation.

Male Fischer-344 rats were exposed to target concentrations of 0, 50, 150 or 450 ppm ethylene oxide (EO) for 6 h/day for 1 or 3 days. Blood was removed by cardiac puncture and cultured in the presence of 5-bromodeoxyuridine for sister-chromatid exchange (SCE) and chromosome-breakage analyses. EO caused a concentration-dependent increase in SCE following both 1 and 3 days of exposure, and the effects appeared to be additive. No significant dose-dependent increase in chromosome breakage was observed, and reasons for this apparent discrepancy with other published data are discussed. These findings are consistent with the occurrence of elevated SCE frequencies in occupationally-exposed workers, and the results demonstrate for the first time that SCE can be detected in cultured lymphocytes of rodents following inhalation exposures.

Arsenic can mediate skin neoplasia by chronic stimulation of keratinocyte-derived growth factors.

Although numerous epidemiological studies have shown that inorganic arsenicals are human skin carcinogens, there is currently no accepted mechanism for its action or an established animal model for its study. We observed increased mRNA transcripts and secretion of keratinocyte growth factors, including granulocyte macrophage-colony stimulating factor (GM-CSF) and transforming growth factor-alpha (TGF-alpha) and the proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) in primary human epidermal keratinocytes cultured in the presence of low micromolar concentrations of sodium arsenite. Total cell numbers, as well as c-myc expression and incorporation of [3H]thymidine, both indicators of cell proliferation, were also elevated in keratinocyte cultures treated with sodium arsenite. As an in vivo model, the influence of arsenic on mouse skin tumor development was studied in transgenic TG.AC mice which carry the v-Ha-ras oncogene, and can serve as a genetically initiated model for skin carcinogenesis. Following low-dose application of 12-O-tetradecanoyl phorbol-13-acetate (TPA), a marked increase in the number of skin papillomas occurred in transgenic mice receiving arsenic in the drinking water as compared to control drinking water. Papillomas did not develop in arsenic-treated transgenic mice that had not received TPA or arsenic-treated wild-type FVB/N mice, suggesting that arsenic is neither a tumor initiator or promoter but rather an enhancer. Injection of anti-GM-CSF antibodies following application of TPA in transgenic mice reduced the number of papillomas. Consistent with that observed in human keratinocyte cultures, increases in GM-CSF and TGF-alpha mRNA transcripts were found within the epidermis of arsenic-treated mice when compared to controls within 6 weeks of treatment. These results suggest that arsenic enhances papilloma development via the chronic stimulation of keratinocyte-derived growth factors and represents the first example of a chemical carcinogen that acts in this manner. These studies suggest that in vitro studies with human keratinocyte cultures examined in conjunction with TG.AC transgenic mice can provide a useful model for examining the tumor enhancing properties of environmental chemicals.

Chemical induction of interleukin-8, a proinflammatory chemokine, in human epidermal keratinocyte cultures and its relation to cytogenetic toxicity.

Tumor promoters, proinflammatory cytokines, endotoxins, and protein synthesis inhibitors can modulate cell cycle kinetics of various cell types, stimulate production of reactive oxygen species, and induce keratinocytes to produce interleukin-8 (IL-8), a potent chemotactant for polymorphonuclear neutrophils and T lymphocytes. The aim of this study was to determine whether perturbations of cytogenetic responses correlated with the induction of IL-8 expression. Cultures of primary human keratinocytes were grown in serum-free medium with 5 mumol/L bromodeoxyuridine to label DNA and exposed either to phorbol-13-myristate-12-acetate (PMA) (0.0001-100 ng/ml), cycloheximide (CHX) (0.01-50 micrograms), lipopolysaccharide (0.1-100 micrograms/ml), tumor necrosis factor-alpha (TNF alpha) (3.13-50 ng/ml), or interleukin-1 alpha (IL-1 alpha) (1-182 pg/ml). Metaphase chromosome preparations were stained by a fluorescence-plus-Giemsa technique to differentiate sister chromatids. For IL-8 production, keratinocytes were grown to 70% confluency and then exposed to chemicals for 24 h. Immunoreactive IL-8 was quantitated from the supernatants by ELISA. With the exception of benzo(a)pyrene used as a positive control, none of the agents induced sister chromatid exchanges. However, PMA and TNF alpha induced IL-8 production that coincided with significant cell cycle inhibition. IL-1 alpha had no effect on cytogenetic endpoints, yet stimulated a 6.3-fold increase in IL-8. CHX inhibited cell cycle progression and mitotic activity at concentrations that were 200 times lower than required for IL-8 induction; however, puromycin (0.31-10 micrograms/ml), another protein synthesis inhibitor, did not induce IL-8. At all concentrations tested, TNF alpha reduced the mitotic index by approximately 45%, slowed cell cycle progression by approximately 3.5 h, and induced a flat, albeit large, IL-8 response at concentrations > or = 12.5 ng/ml. These agent-specific response patterns suggest that induction of IL-8 production is not always the inevitable result of cell cycle perturbations or genetic damage.