Induction of chromosomal aberrations and sister chromatid exchanges by propane sultone in human lymphocytes in vitro

Genotoxicological investigation of 1,3-propane sultone was undertaken using human lymphocyte cultures as the test system. Concentrations greater than 1 mM induced appreciable chromosomal aberrations, which increased proportionally to the dose or the duration of treatment. It was also observed that treatment administered at 45 degrees C produced almost 3 times more abnormal cells than at 37 degrees C. The chemical also produced a significantly higher frequency of SCEs.     

The frequency of sister chromatid exchanges in cultured human peripheral blood lymphocyte treated with metronidazole in vitro

5-Nitroimidazoles are a group of antiprotozoal and antibacterial agents. Thanks to their antimicrobial activity, these chemotherapeutic agents inhibit the growth of both anaerobic bacteria and certain anaerobic protozoa. One of the useful drugs used in the treatment of infections caused by Trichomonas vaginalis, Entamoeba histolytica, and Giardia lamblia is metronidazole (MTZ). The mutagenicity of metronidazole [1-(hydroxyethyl)-2-methyl-5-nitroimidazole] has been previously shown in different prokaryotic systems but not in eukaryotic systems. The objective of this study is to determine the mutagenic and cytotoxic effects of MTZ at plasma concentration and higher in vitro. In this study, we evaluated the mutagenicity and cytotoxicity of MTZ in cultured human peripheral blood lymphocytes. Doses were selected according to plasma concentration of drug. End points analyzed included mitotic index (MI), replication index (RI), and sister chromatid exchange (SCE). An analysis of variance test (ANOVA) was performed to evaluate the results. A significant decrease (p < 0.001) in MI and RI as well as an increase in SCE frequency (p < 0.001) was observed compared with control cultures. Our results indicate the genotoxic and cytotoxic effect of MTZ in cultured human peripheral blood lymphocytes at plasma concentrations slightly higher than encountered therapeutically.     

Influence of the level of cytosolic epoxide hydrolase on the induction of sister chromatid exchanges by trans-beta-ethylstyrene 7,8-oxide in human lymphocytes

trans-beta-Ethylstyrene 7,8-oxide, a substrate of cytosolic epoxide hydrolase, and 4-fluorochalcone oxide, an inhibitor of this enzyme, were investigated on induction of sister chromatid exchanges (SCE) in human lymphocytes. Both epoxides enhanced the frequency of SCE. 4-Fluorochalcone oxide at low concentration (2.5 microM) inhibited cytosolic epoxide hydrolase activity towards trans-beta-ethylstyrene 7,8-oxide in lymphocytes by 74% and had no effect on glutathione transferase activity using this substrate. At this concentration it did not induce SCE itself, but it potentiated the effect of trans-beta-ethylstyrene 7,8-oxide several fold. In lymphocytes from different subjects, the number of SCE induced by a low concentration of trans-beta-ethylstyrene 7,8-oxide correlated negatively with the individual cytosolic epoxide hydrolase activity (r = -0.72; -0.73 in two series of experiments). The number of SCE induced by a high concentration of trans-beta-ethylstyrene 7,8-oxide did not correlate with cytosolic epoxide hydrolase activity (r = 0.004; -0.24), but a negative correlation was found with glutathione transferase activity (r = -0.50). This finding is consistent with the results of biochemical studies in lymphocytes in which we determined the relative contribution of cytosolic epoxide hydrolase and glutathione transferase to the metabolism of trans-beta-ethylstyrene 7,8-oxide at varying substrate concentrations. The study demonstrates that the level of genotoxic effects induced in human lymphocytes is influenced by the individual level of detoxifying enzymes. At low concentrations, cytosolic epoxide hydrolase was more important than glutathione transferase activity.     

Induction of sister chromatid exchanges and chromosome aberrations in vivo in Etroplus suratensis (Bloch) following exposure to organophosphorus pesticides

The genotoxic potential of methyl parathion and phosphamidon, two commercial formulations of organophosphorus pesticides, was evaluated through induction of sister chromatid exchanges (SCE) and chromosome aberrations in fish gill tissues. Fishes exposed to the medium containing 0.05, 0.1 and 0.2 ppm of methyl parathion or 0.5, 1.0, and 2.0 ppm of phosphamidon for a duration of 96 h revealed significant increase in the number of SCE and chromosome aberrations against control values. This demonstrates the feasibility of the fish in vivo system in detecting genotoxic potential of pollutants.     

Induction of DNA strand breaks, DNA-protein crosslinks and sister chromatid exchanges by arsenite in a human lung cell line.

Based on in vitro studies, several modes of action for arsenic have been suggested, although the mechanisms responsible for arsenic carcinogenesis have not been well established. In our previous study a dose-dependent increment in DNA migration was detected at low doses of sodium arsenite, but at higher dose levels a reduction in the migration was observed, suggesting the induction of DNA adducts. In order to confirm this hypothesis we performed the experiments considering other parameters and modifications of the standard alkaline comet assay. Additionally, the induction of sister chromatid exchanges was evaluated. The present study showed the induction by sodium arsenite of single strand breaks and DNA-protein adducts assessed by comet assay as well as of sister chromatid exchanges in the human lung fibroblast cell line MRC-5. The standard alkaline comet assay also revealed, at the highest arsenic concentration tested, a reduction in all the considered parameters in relation to untreated cells and the other doses. On the other hand, the incubation with proteinase K induced a dose-dependent increment in DNA migration as a consequence of the release of proteins joined to the DNA. Thus, sodium arsenite was able to induce both DNA-strand breaks and protein-DNA adducts in arsenic exposed MRC-5 cells, depending on the concentrations of arsenic salts tested.     

Polymorphism of glutathione conjugation of methyl bromide,ethylene oxide and dichloromethane in human blood: Influence on the induction of sister chromatid exchanges (SCE) in lymphocytes

A hitherto unknown glutathione-S-transferase in human erythrocytes displays polymorphism: three quarters of the population (conjugators) possess, whereas one quarter (non-conjugators) lack this specific activity. A standard method for the identification of conjugators and non-conjugators with the use of methyl bromide and gas chromatography (head space technique) is described. Three substrates of the polymorphic enzyme, methyl bromide, ethylene oxide and dichloromethane (methylene chloride), were incubated in vitro with individual whole blood samples of conjugators and non-conjugators. All three substances led to a marked increase of sister chromatid exchanges (SCE) in the lymphocytes of the non-conjugators but not in those of conjugators. A protective effect of the glutathione-S-transferase activity in human erythrocytes for the cytogenetic toxicity of these chemicals in vitro is thus confirmed. Since the enzyme activity is not found in erythrocytes of laboratory animals, species extrapolations for risk assessment of methyl bromide, ethylene oxide and dichloromethane should be reconsidered.     

In vitro and in vivo effects of melatonin on sister chromatid exchange in human blood lymphocytes exposed to hypoxia

Objective: Many studies have shown that melatonin (MLT) has an anti-genotoxic effect in various tissues and cell lines. The aim of this study was to investigate the anti-genotoxic effect of MLT on normal human peripheral lymphocytes by assessing sister chromatid exchange (SCE) in vitro and in vivo. Materials and methods: Cells were treated with 50 and 200?μM of MLT. The human volunteers (n?=?20) for the in vivo study were administered a single dose of 3?mg MLT daily for 2 weeks. After sufficient time for its clearance, 1.5?mg of MLT daily was then administered to the same volunteers at same the period. Results: Our results demonstrated the anti-genotoxic effect of MLT in human blood lymphocyte in vitro and in vivo. In vitro, hypoxia increased the SCE frequency compared to the control and both doses of MLT significantly decreased the SCE frequency in the hypoxic cells (p?<?0.001). In vivo, oral administration of 3?mg MLT significantly increased the frequency of SCE, yet a small increase of SCE by hypoxia was found. Oral administration of 1.5?mg MLT showed no DNA damage but it had an anti-genotoxic effect. Discussion and conclusion: MLT may prove useful for reducing the genotoxic effects of hypoxia in peripheral lymphocytes and suggest its possible role for ischemic diseases.     

Promutagen activation of triazine herbicides metribuzin and ametryn through Vicia faba metabolism inducing sister chromatid exchanges in human lymphocytes in vitro and in V. faba root tip meristems.

The aim of our study was the induction of sister chromatid exchanges (SCE) in human lymphocytes in vitro and in root tip meristems of Vicia faba to evaluate the genotoxic effects of metribuzin and ametryn. Direct treatments of these herbicides on human lymphocytes in vitro applied 24 h after the beginning of culture did not induce SCE; however, they showed a cytotoxic effect in the cultures expressed as cellular death. On the contrary, when extracts of V. faba roots, treated for 4 h with metribuzin and ametryn (in vivo activation), were added to the lymphocyte cultures, SCEs were significantly induced with an asymptotic response. Negative responses appeared with the in vitro assays, in which metribuzin and ametryn were added directly to the 48 h lymphocyte cultures for 4 h. Nevertheless, in treatments in which the S10 metabolic mix was added, the SCE frequencies were significantly different to the control, although a concentration-response relationship was only observed with metribuzin. The results showed that both herbicides needed the V. faba metabolism to produce SCE in human lymphocyte cultures. Metribuzin and ametryn applied to V. faba root tip meristems for 4 h increased SCE frequency significantly, and a concentration-response relationship was observed with both herbicides.     

Evidence for induction of germinal aplasia in male rats by environmental exposure to boron

In view of recent reports of adverse effects of environmental boron on testicular function, we have studied some biochemical, morphological, and physiologic aspects of gonadal function in adult male rats exposed to increased levels of dietary boron. The experimental animals were fed 500, 1000, and 2000 ppm of boron added as borax to standard laboratory chow for 30 to 60 days. Fertility, testicular, and epididymal histology; specific activities of testicular and prostatic enzymes; plasma concentrations of FSH, LH, and testosterone, and boron content of plasma and testes were evaluated. Some of the rats treated with the highest dose were followed for up to 1 year following cessation of boron exposure. In both 30- and 60-day feeding studies, male rats receiving 500 ppm suffered no significant adverse effects. In contrast, male rats receiving 1000 and 2000 ppm of boron displayed a significant loss of germinal elements, decrease in seminiferous tubular diameter, and accumulation of testicular boron. The testicular atrophy was greatest at the highest dose, and depletion of germ cells was complete after 60 days of exposure. These morphological changes were associated with reduction of specific activities of hyaluronidase, sorbitol dehydrogenase, and lactate dehydrogenase isozyme-X, markers of postmeiotic germ cells. In contrast, specific enzyme activities of glyceraldehyde 3-phosphate and malate dehydrogenase were significantly elevated above control values, an effect probably of the relative enrichment of Sertoli cells and spermatogonia. The germinal depletion was associated with a significant increase in plasma FSH concentration, a variable increase in plasma LH levels (not significant), and normal concentrations of plasma testosterone. The increased plasma FSH and germinal cell depletion were time- and dose-dependent. Serial mating studies demonstrated reduced fertility without change in copulatory behavior. The duration of reduced fertility was greater at the higher dose and the longer duration of exposure. Germinal aplasia, elevated FSH, and infertility persisted for at least 8 months following cessation of boron exposure at the highest dose. These data suggest that boron accumulation in testes is associated with progressive germ cell depletion and that the testicular lesion can persist long after toxic exposure to boron has occurred. Further long-term studies are necessary to determine whether testicular dysfunctions can occur after prolonged exposure to levels of boron which prevail in some areas of the environment.     

Saint John's wort: an in vitro analysis of P-glycoprotein induction due to extended exposure.

1. Chronic use of Saint John's wort (SJW) has been shown to lower the bioavailability for a variety of co-administered drugs including indinavir, cyclosporin, and digoxin. Decreases in intestinal absorption through induction of the multidrug resistance transporter, P-glycoprotein (P-gp), may explain decreased bioavailability. 2. The present study characterized the response of P-gp to chronic and acute exposure of SJW and hypericin (HYP, a presumed active moiety within SJW) in an in vitro system. Experiments were performed with 3 to 300 microg ml(-1) of methanol-extracted SJW and 0.03 to 3 microM HYP, representing low to high estimates of intestinal concentrations. 3. In induction experiments, LS-180 intestinal carcinoma cells were exposed for 3 days to SJW, HYP, vehicle or a positive control (ritonavir). P-gp was quantified using Western blot analysis. P-gp expression was strongly induced by SJW (400% increase at 300 microg ml(-1)) and by HYP (700% at 3 microM) in a dose-dependent fashion. Cells chronically treated with SJW had decreased accumulation of rhodamine 123, a P-gp substrate, that was reversed with acute verapamil, a P-gp inhibitor. Fluorescence microscopy of intact cells validated these findings. In Caco-2 cell monolayers, SJW and HYP caused moderate inhibition of P-gp-attributed transport at the maximum concentrations tested. 4. SJW and HYP significantly induced P-gp expression at low, clinically relevant concentrations. Similar effects occurring in vivo may explain the decreased bioavailability of P-gp substrate drugs when co-administered with SJW.     

Oxidative stress resulting from exposure of a human salivary gland cells to paraoxon: An in vitro model for organophosphate oral exposure

Organophosphate (OP) compounds are used as insecticides, acaricides, and chemical agents and share a common neurotoxic mechanism of action. The biochemical alterations leading to many of the deleterious effects have been studied in neuronal cell lines, however, non-neuronal toxic effects of OPs are far less well characterized in vitro, and specifically in cell lines representing oral routes of exposure. To address this void, the human salivary gland (HSG) cell line, representing likely interactions in the oral cavity, was exposed to the representative OP paraoxon (PX; O,O-diethyl-p-nitrophenoxy phosphate) over a range of concentrations (0.01–100 μM) and analyzed for cytotoxicity. PX induced cytotoxicity in HSG cells at most of the exposure concentrations as revealed by MTT assay, however, the release of LDH only occurred at the highest concentration of PX tested (100 μM) at 48 h. Slight increases in cellular ATP levels were measured in PX-exposed (10 μM) HSG cells at 24 h. Exposing HSG cells to 10 μM PX also led to an increase in DNA fragmentation prior to loss of cellular membrane integrity implicating reactive oxygen species (ROS) as a trigger of toxicity. The ROS genes gss, gstm2, gstt2 and sod2 were upregulated, and the presence of superoxide following 10 μM PX exposure was determined via dihydroethidium fluorescence studies further implicating PX-induced oxidative stress in HSG cells.     

Cryopreserved primary hepatocytes as a constantly available in vitro model for the evaluation of human and animal drug metabolism and enzyme induction

The use of primary hepatocytes is now well established for both studies of drug metabolism and enzyme induction. Cryopreservation of primary hepatocytes decreases the need for fresh liver tissue. This is especially important for research with human hepatocytes because availability of human liver tissue is limited. In this review, we summarize our research on optimization and validation of cryopreservation techniques. The critical elements for successful cryopreservation of hepatocytes are (1) the freezing protocol, (2) the concentration of the cryoprotectant [10% dimethyl-sulfoxide (DMSO)], (3) slow addition and removal of DMSO, (4) carbogen equilibration during isolation of hepatocytes and before cryopreservation, and (5) removal of unvital hepatocytes by Percoll centrifugation after thawing. Hepatocytes of human, monkey, dog, rat, and mouse isolated and cryopreserved by our standard procedure have a viability > or = 80%. Metabolic capacity of cryopreserved hepatocytes determined by testosterone hydroxylation, 7-ethoxyresorufin-O-de-ethylase (EROD), 7-ethoxycoumarin-O-deethylase (ECOD), glutathione S-transferase, UDP-glucuronosyl transferase, sulfotransferase, and epoxide hydrolase activities is > or = 60% of freshly isolated cells. Cryopreserved hepatocytes in suspension were successfully applied in short-term metabolism studies and as a metabolizing system in mutagenicity investigations. For instance, the complex pattern of benzo[a]pyrene metabolites including phase II metabolites formed by freshly isolated and cryopreserved hepatocytes was almost identical. For the study of enzyme induction, a longer time period and therefore cryopreserved hepatocyte cultures are required. We present a technique with cryopreserved hepatocytes that allows the induction of testosterone metabolism with similar induction factors as for fresh cultures. However, enzyme activities of induced hepatocytes and solvent controls were smaller in the cryopreserved cells. In conclusion, cryopreserved hepatocytes held in suspension can be recommended for short-term metabolism or toxicity studies. Systems with cryopreserved hepatocyte cultures that could be applied for studies of enzyme induction are already in a state allowing practical application, but may be further optimized.     

Airway injury in an in vitro human epithelium-fibroblast model of diacetyl vapor exposure: diacetyl-induced basal/suprabasal spongiosis

Inhalation exposure to diacetyl (DA) is associated with obliterative bronchiolitis (OB) in workers and induces OB-like fibrotic airway lesions in rats. The pathogenesis of OB is poorly understood in part due to complex interactions between airway epithelial, mesenchymal and blood-derived inflammatory cells. DA-induced airway toxicity in the absence of recruited-inflammatory/immune cells was characterized using an air-liquid interface (ALI) model consisting of human airway epithelium with (Epi/FT) and without (Epi) a mesenchymal component. ALI cultures were exposed to 25?mM DA-derived vapors (using vapor cups) for 1?h on day 0, 2 and 4. In some experiments, the tissues were exposed to 2,3-hexanedione (Hex) which is structurally-similar, but much less fibrogenic than DA. Lactate dehydrogenase activity and day 6 histopathologic changes associated with epithelial injury, including basal/suprabasal spongiosis, were increased following exposure of Epi/FT tissues to DA but not control or Hex vapors. IL-1a, IL-6, IL-8, sIL-1Ra, TGFa, MCP-3 and TNFa proteins were increased following DA exposure of Epi/FT tissues; only IL-1a, IL-8, sIL-1Ra and TGFa were increased following exposure of Epi tissues. MMP-1, MMP-3 and TIMP-1 proteins were increased following DA exposure of Epi/FT tissues; whereas MMP-2, MMP-7 and TIMP-2 were decreased, and production was largely dependent upon the presence of sub-epithelial stromal matrix/fibroblasts. Hex-induced protein changes were minimal. This in vitro study demonstrated that exposure of human airways to DA vapors induced epithelial injury (with the histopathologic feature of basal/suprabasal spongiosis) and increased release of pro-inflammatory and pro-fibrotic cytokines/chemokines as well as MMPs/TIMPs in the absence of recruited-inflammatory cells.     

Compensatory nicotine self-administration in rats during reduced access to nicotine: an animal model of smoking reduction

The ability of smoking reduction (e.g., decreasing cigarettes per day) to produce significant reductions in toxin exposure is limited by compensatory increases in smoking behavior. Characterizing factors contributing to the marked individual variability in compensation may be useful for understanding this phenomenon. The goal of the current study was to develop an animal model of smoking reduction and to begin to examine potential behavioral and pharmacokinetic contributors to compensation. Rats trained for nicotine self-administration (NSA) in unlimited access sessions were exposed to a progressive decrease in duration of access to nicotine from 23-hr/day to 10-, 6-, and 2-hr/day. Following a return to 23 hr/day access and extinction, single-dose nicotine pharmacokinetic parameters were determined. Rats exhibited a reduction in total daily nicotine intake during reduced access to NSA, but decreases in nicotine intake were not proportional to decreases in access duration. Compensatory increases in hourly infusion rate were also observed when access was decreased. The magnitude of compensation differed considerably among animals. Early session infusion rate during baseline was significantly correlated, while nicotine clearance was moderately correlated, with 1 measure of compensation. Infusion rates were transiently increased compared to prereduction levels when unlimited access was restored, and this effect was greatest in animals that had exhibited the greatest levels of compensation. These findings indicate that rats exhibit compensatory increases in NSA during reduced access to nicotine, with substantial individual variability. This model may be useful for characterizing underlying factors and potential consequences of compensatory smoking.     

Prevalidation of a human cornea construct as an alternative to animal corneas for in vitro drug absorption studies

The use of ophthalmic drugs has increased consistently over the past few decades. Currently, most research is conducted using in vivo and ex vivo animal experiments; however, they have many disadvantages, including ethical concerns, high costs, the questionable extension of animal results to humans, and poor standardization. Although several cell culture-based cornea models have been developed, none have been validated and accepted for general use. In this study, a standardized, three-dimensional model of the human cornea (Hemicornea, HC) based on immortalized human corneal cells and cultivated in serum-free conditions was developed for drug absorption studies and prevalidated using compounds with a wide range of molecular characteristics (sodium fluorescein, rhodamine B, fluorescein isothiocyanate-labeled dextran, aciclovir, bimatoprost, dexamethasone, and timolol maleate). The HC model was independently cultured in three different laboratories, and the intralaboratory and interlaboratory reproducibility was analyzed and compared with the rabbit cornea. This analysis showed that the HC has a barrier in the same range as excised animal corneas, although with a higher reproducibility and lower variability. Because of the demonstrated transferability, the HC represents a promising in vitro alternative to the use of ex vivo tissue and offers a well-defined and standardized system for drug absorption studies.     

Interactions between chronic haloperidol treatment and cocaine in rats: an animal model of intermittent cocaine use in neuroleptic treated populations

This experiment investigated the possibility that rats maintained on chronic haloperidol treatment would show increased behavioral responsiveness to cocaine, similar to that observed in human stimulant abusers who are chronically treated with neuroleptics. Thus, the effects on locomotion and stereotyped behavior of intermittent injections of cocaine were investigated in female rats receiving chronic haloperidol treatment. Daily injections of haloperidol (0.2 mg/kg, IP) or vehicle were administered for 6, 12 or 18 days prior to the start of testing with cocaine and were then continued throughout cocaine testing. All rats received four doses of cocaine (0.0, 3.0, 7.5, or 15.0 mg/kg, IP) in random order with an intervening vehicle day between successive drug days. The four dose sequence of cocaine was repeated a total of four times. Initial cocaine administration produced dose dependent increases in locomotion and stereotyped behavior. When the sequence of cocaine doses was repeated, differences among treatment groups emerged. Groups treated with haloperidol exhibited heightened locomotion in response to cocaine and with repeated injections, showed a higher rate of behavioral sensitization than control animals. These differences in the behavioral response to cocaine were maintained for at least 2 months following termination of daily haloperidol treatment. In order to examine the mechanisms underlying this heightened responsiveness to cocaine, apomorphine-induced locomotion (dose range, 0–250 µg/kg, SC) was determined. Regardless of dose, rats treated with haloperidol showed different temporal patterns of locomotion in response to apomorphine suggesting that the increased response to cocaine was related to changes in dopaminergic receptor sensitivity.     

Assessment of alterations in barrier functionality and induction of proinflammatory and cytotoxic effects after sulfur mustard exposure of an in vitro coculture model of the human alveolo-capillary barrier

Acute lung injury after sulfur mustard (SM) inhalation is characterized by massive, localized hemorrhage and alveolar edema, which implies severe disruption of the vascular and distal airway barrier. In this study, we tested a recently established in vitro coculture model of the alveolo-capillary barrier for its applicability to investigate acute toxic effects of SM at the human respiratory unit. The epithelial compartment of cocultures was exposed to varying concentrations of SM (0-1000 microM; t = 30 min). Following exposure, functional and structural barrier integrity of cocultures was monitored over a period of 24 h. A 50% reduction of transbilayer electrical resistance (TER) within 12-24 h after exposure to 300 microM SM and within 8 h after 1000 microM SM revealed a time- and concentration-dependent impairment of barrier functionality, which was associated with structural loss of both cell layers. Subsequent quantification of interleukin (IL)-6 and IL-8 in cell culture supernatants of exposed cocultures showed enhanced liberation of proinflammatory markers. Highest mediator levels were detected after 300 microM SM, with pronounced stimulation in the endothelial compartment. SM-related cytotoxicity was determined by assessing adenylate kinase (AK) release and by quantifying the fraction of DNA-fragmented nuclei using terminal deoxynucleotidyl transferase-mediated DNA nick-end labeling (TUNEL) and nuclear Hoechst staining. Both methods exposed a concentration-dependent increase of SM-mediated cytotoxic effects with high effects on endothelial cells. We conclude that the described in vitro model reflects important characteristics of SM-mediated acute lung injury in vivo and thus can be used to explore involved pathophysiological pathways.