Genotoxicity and apoptosis in Drosophila melanogaster exposed to benzene, toluene and xylene: attenuation by quercetin and curcumin.

Monocyclic aromatic hydrocarbons (MAHs) such as benzene, toluene and xylene are being extensively used for various industrial and household purposes. Exposure to these hydrocarbons, occupationally or non-occupationally, is harmful to organisms including human. Several studies tested for toxicity of benzene, toluene and xylene, and interestingly, only a few studies looked into the attenuation. We used Drosophila model to test the genotoxic and apoptotic potential of these compounds and subsequently evaluated the efficiency of two phytochemicals, namely, quercetin and curcumin in attenuating test chemical induced toxicity. We exposed third instar larvae of wild type Drosophila melanogaster (Oregon R+) to 1.0-100.0 mM benzene, toluene or xylene, individually, for 12, 24 and 48 h and examined their apoptotic and genotoxic potential. We observed significantly (P<0.001) increased apoptotic markers and genotoxicity in a concentration- and time-dependent manner in organisms exposed to benzene, toluene or xylene. We also observed significantly (P<0.001) increased cytochrome P450 activity in larvae exposed to test chemicals and this was significantly reduced in the presence of 3',4'-dimethoxyflavone, a known Aryl hydrocarbon receptor (AhR) blocker. Interestingly, we observed a significant reduction in cytochrome P450 activity, GST levels, oxidative stress parameters, genotoxic and apoptotic endpoints when organisms were exposed simultaneously to test chemical along with quercetin or curcumin. The study further suggests the suitability of D. melanogaster as an alternate animal model for toxicological studies involving benzene, toluene and xylene and its potential in studying the protective role(s) of phytochemicals.

Mechanism of flavin transfer and oxygen activation by the two-component flavoenzyme styrene monooxygenase.

Styrene monooxygenase (SMO) from Pseudomonas putida S12 is a two-component flavoenzyme composed of the NADH-specific flavin reductase, SMOB, and FAD-specific styrene epoxidase, SMOA. Here, we report the cloning, and expression of native and histidine-tagged versions of SMOA and SMOB and studies of the flavin transfer and styrene oxygenation reactions. In the reductive half-reaction, SMOB catalyzes the two-electron reduction of FAD with a turnover number of 3200 s(-1). Single turnover studies of the reaction of reduced SMOA with substrates indicate the formation of a stable oxygen intermediate with the absorbance characteristics of a flavin hydroperoxide. Based on the results of numerical simulations of the steady-state mechanism of SMO, we find that the observed coupling of NADH and styrene oxidation can be best explained by a model, which includes both the direct transfer and passive diffusion of reduced FAD from SMOB to SMOA.

Substrate inhibition kinetics for toluene and benzene degrading pure cultures and a method for collection and analysis of respirometric data for strongly inhibited cultures.

We present an evaluation of the qualitative and quantitative effects that high concentrations of benzene and toluene have on the growth rate of several pure cultures that use these compounds as their sole carbon and energy source. The cultures employed were five widely studied environmental isolates: Pseudomonas putida F1, P. putida mt2, P. mendocina KR, Ralstonia pickettii PKO1, and Burkholderia cepacia G4. Three cultures degraded toluene following a pattern consistent with the kinetic model of Wayman and Tseng (1976) while the other two followed a modification of this model introduced by Alagappan and Cowan (2001). The pattern followed for benzene degradation was different than that for toluene degradation for all four capable pure cultures and consistent with that described by the model of Luong (1987). Mechanisms of substrate inhibition and solvent toxicity are discussed, used to conceptually evaluate the reasons for the differences in inhibition behavior, and used to support a call for more widespread use of the empirical, terminal substrate concentration inhibition models employed here. We also present the methodology developed to overcome a limitation commonly encountered when attempting to collect oxygen uptake data for use in quantifying substrate inhibition kinetics. The experimental method was effective for use in the collection of high quality data and the substrate inhibition models most useful in representing the growth of bacteria on these solvents are those that show a complete loss of activity at high concentration rather than the more popular asymptotic inhibition models.

Application of gas chromatography-tandem mass spectrometry to the analysis of inhibition of dimerisation of tributylphosphate under radiolysis. Identification of isomeric tributylphosphate-alkylbenzene inhibitor coupling products.

Tributylphosphate (TBP), solvent used as extractant for reprocessing spent nuclear fuel, can dimerise under radiolysis. This occurs by radical radical recombination, leading to 10 isomeric dimers (TBP-TBP). These species are complexation agents and are responsible of fission product retention in the organic phase that increases the solvent degradation. In order to limit their formation two free radical inhibitors (In), isopropyl and 1,4-diisopropylbenzenes, were used. These additives reduce by about 50% the concentration of TBP-TBP dimers but this reduction is not strictly followed by TBP regeneration as mixed coupling products from TBP and inhibitor are detected. By using GC-MS-MS and selectively deuterated compounds, the identification of these different isomers (TBP-In) has been realised. From these identifications and from the analysis of the proportion of the different isomers, the major primary TBP radical generated under radiolysis was determined.

Dexamethasone ameliorates oxidative DNA damage induced by benzene and LPS in mouse bone marrow.

Mice were grouped to receive vehicle, dexamethasone (DEX), lipopolysaccharide (LPS), benzene (BZ, 200 mg/kg) and combinations: LPS + DEX, BZ + DEX, LPS + BZ, LPS + DEX + BZ. The DNA damage in bone marrow cells from BZ group was enhanced 2.8-fold measured by nuclear 8-hydroxy-2 '-deoxyguanosine (8-oxodG) and 1.4-fold measured by Comet score (index of DNA breaks) (p < 0.05). In the BZ + DEX group, 8-oxodG level and the Comet score were lowered to 65% and 76% respectively of that in the BZ group (p < 0.05). The BZ + LPS caused a 3.9-fold increase in 8-oxodG and a 1.6-fold increase in the Comet score (p < 0.05). The LPS + DEX + BZ lowered 8-oxodG level and the Comet score to 50% and 78% of the values in the LPS + BZ group, respectively (p < 0.05). Nitrate/nitrite levels in serum were higher after BZ + LPS treatment than after all other treatments. Both 8-oxodG level and the Comet scores were correlated to the serum nitrate/nitrite level across all the treatments (r = 0.55, p < 0.01 and r = 0.69, p < 0.01, respectively). In bone marrow cells the 8-oxodG correlated with the Comet scores (r = 0.80, p < 0.01). We conclude that DEX administration can reduce the DNA damage from BZ treatment and from the combination of BZ and LPS. The correlation of DNA damage with nitrate/nitrite indicates the possible involvement of reactive nitrogen species (RNS) in the interaction between BZ and the inflammatory reaction stimulated by LPS. The 8-oxodG determination is more sensitive than strand break analysis by the Comet assay in bone marrow in vivo in mice for measuring the BZ-induced DNA damage.

Antigenotoxic effects of cimetidine against benzene induced micronuclei in mouse bone marrow erythrocytes.

An in vivo micronucleus assay using Balb/C male mice was used to examine antigenotoxic effects of cimetidine (CM) on benzene (BZN) induced genotoxic effects. CM not only has therapeutic and immunomudolatory role, but it has also been shown to protect bone marrow stem cells from radiation induced clastogenic effects. Therefore, in the present study we attempt to investigate the protective effects and possible mechanisms involved in the effects of CM. An 8-week-old male Balb/C mice (22+/-4 g weight) were treated with different doses of BZN (400, 600 and 800 mg/kg body weight), i.p. and sampled at 24, 48 and 72 h after treatment by cervical dislocation. Various doses of CM (10, 15, 30 mg/kg) were used in association with BZN and 1-2 h prior to BZN treatment. Results show that BZN effectively induced micronuclei in polychromatic erythrocytes (PCEs). Application of CM led to a significant reduction of micronuclei in PCEs, i.e. 2-fold after 10 mg/kg and 3-fold after 30 mg/kg CM treatment. Results also indicate CM was more effective when used in combination with BZN. Therefore, results indicate that CM could reduce clastogenic effects of BZN. Although further investigations are needed to reveal the mechanistical background behind the effect, the most probable mechanism involved might be free radical scavenging. This mechanism might be associated with amplification of glutathione system and cytochrome P-450 inhibition.

Immuno-, phagocytosis-modulating, and antitoxic properties of amino acids and peptide preparations.

The influence of amino acids, their mixtures, and peptides on the immune response, phagocytosis in vitro, and in CBA mice, and broiler chickens, as well as on in vitro ability of listed preparations to protect animals' splenocytes from toxic action of benzene and aflatoxin B1 were studied. It was shown that amino acids (Asp, Glu, Val, Trp), amino acid mixtures (cerebrolysine, levamine, aviamine), and the dipeptides GluTrp and LysAsp stimulate the immune response to SRBC at subcutaneous and peroral application. The peptides thymopentin, thymosin alpha-1, and peptide mixtures (thymosin fraction 5, thymalin) stimulate the immune response only at the site of subcutaneous injections. Lys, Tyr, and bursin (LysHisGly-amide), regardless of the mode of application, do not change, but Arg inhibits the immune response. None of the preparations studied change the immune response to Viantigen. Levamine, cerebrolysine, and aviamine are immunoreactive only at low doses (6.5 x 10(2)-6.5 x 10(-8) mg/kg). At a dose of 65 mg/kg these preparations lose immunostimulating properties. Amino acids (6.5 x 10(-2) mg/kg), which stimulate, rather than influence or inhibit the immune response, enhance phagocytosis of S. aureus by granulocytes, regardless of mode of application. Levamine and cerebrolysine in the range of doses of 6.5 x 10(-2)-6.5 x 10(-6) mg/kg do not influence phagocytosis; at a dose of 65 mg/kg, phagocytosis is enhanced. Aviamine stimulates phagocytosis as well at low and at high doses. The ability of preparations to protect in vitro at a low concentration (1.3 x 10(-3) mg/ml) murine or chickens' splenocytes from toxic action of benzene and aflatoxin B1 (at 1:1000 dilution) does not correlate with their action on the immune response and phagocytosis. The protective actions revealed by the following preparations include: (a) stimulating the immune response and phagocytosis (Glu, Asp, Trp, amino acid mixture aviamine, the dipeptides LysAsp, GluTrp, the peptide mixtures thymalin, thymosin fraction 5); (b) enhancing the immune response but not influencing phagocytosis (Met, levamine, cerebrolysine); (c) influencing neither the immune response nor phagocytosis (Gly, Ile). At the same time those preparations (Lys, Arg) that stimulate phagocytosis but influence the immune response in a different way (Lys does not influence; Arg suppresses the response) are inert as antitoxic agents. Antitoxic properties of amino acid preparations levamine, cerebrolysine, and aviamine retain as well in the assays at a rather large (1.3 mg/ml) concentration like their phagocytosis-stimulating properties.

A morphological analysis of the short-term effects of benzene on the development of the hematological cells in the bone marrow of mice and the effects of interleukin-1 alpha on the process.

Chronic exposure of humans to benzene (BZ), a widely used industrial chemical and a ubiquitous environmental pollutant, causes aplastic anemia and acute myeloid leukemia. The purpose of the studies reported here was to determine whether the observed depression of bone marrow (BM) cellularity in mice administered benzene was reflected in a suppression of development of all of the hematopoietic lineages and to confirm the ability of interleukin-1 alpha (IL-1 alpha) to prevent BZ-induced BM cell depression. We report that BZ, administered twice per day for 2 days to C57B1/6J mice at a dose of 600 mg/kg body weight, caused a significant depression of the total number of nucleated BM cells per femur when measured on day 3. The observed depression reflects a complex situation that represents the net effect of a decrease in the total number of cells of the lymphocytic and erythroid lineages, along with an increase in the number of intermediate and terminally differentiated cells of the granulocytic lineage. An experiment to monitor the effects of BZ over a 7-day period showed a progressive depressive effect on the lymphocytes and an initial depression of the erythroid cells at day 3 that remained constant until day 7. Conversely, the numbers of intermediate and terminally differentiated granulocytes progressively increased over the 7 days. The BM appeared to recover from the depressive effects of BZ immediately upon cessation of exposure, as the number of nucleated BM cells began to rise by day 5 and was equal to that of the control group by day 7. The results expand our earlier finding (Renz and Kalf 1991) that the overall depression of BM cellularity occurs because of an inability of the stromal fibroblast to produce colony-stimulating factors essential for stem and progenitor cell survival. This results from inhibition by the BZ metabolite, hydroquinone (HQ), of the processing of pre-IL-1 alpha to the mature cytokine.(ABSTRACT TRUNCATED AT 250 WORDS)

Prevention of benzene-induced myelotoxicity and prostaglandin synthesis in bone marrow of mice by inhibitors of prostaglandin H synthase.

Administration of benzene to mice causes bone marrow toxicity and elevations in prostaglandin E2 (PGE2), a negative regulator of myelopoiesis. In these experiments, benzene (400 mg/kg; 2 x/day for 2 days) administered to DBA/2 or C57Bl/6 mice decreased bone marrow cellularity and myeloid progenitor cell development (measured as colony-forming units per femur) by 40%. When inhibitors of the cyclooxygenase component of prostaglandin H synthase (PHS) (either indomethacin, 2 mg/kg; aspirin, 50 mg/kg; meclofenamate, 4 mg/kg) were coadministered with benzene, myelotoxicity and the elevation in bone marrow PGE level were prevented. Additionally, when indomethacin (1 microM) was added to cultures of bone marrow cells from benzene-treated mice, myeloid progenitor cell development was the same as the controls. The doses of indomethacin used had no affect on the hepatic conversion of benzene to its major metabolite, phenol. Using purified PHS, indomethacin (10 microM) inhibited the arachidonic acid-dependent oxidation of hydroquinone to p-benzoquinone, a putative reactive metabolite of benzene. Indomethacin (10 microM) had no effect on the H2O2-driven oxidation of hydroquinone catalysed by either PHS-peroxidase or myeloperoxidase. Coadministration of the benzene metabolites, phenol and hydroquinone, has been reported previously to reproduce the myelotoxicity of benzene. In our studies, phenol and hydroquinone (50 mg/kg each; 2 x/day for 2 days) decreased bone marrow cellularity by 40%; however, coadministration of indomethacin (2 mg/kg) or meclofenamate (4 mg/kg) with these metabolites did not prevent the decrease in bone marrow cell number. Our results implicate marrow PHS in mediating the short-term myelotoxicity of benzene.

Modulation of benzene toxicity by polyinosinic-polycytidilic acid, an interferon inducer.

Repeated intraperitoneal administration of benzene (1.0 ml/kg body wt.) for 3 days produced leucopenia, lymphocytopenia and significantly decreased body wt. (P less than 0.001) and organ weights of thymus (P less than 0.001) and spleen (P less than 0.001) in female albino rats. Total iron content, lipid peroxidation and superoxide dismutase activity of the liver and bone marrow were significantly increased as a result of benzene exposure. Low molecular weight (LMW) bleomycin detectable iron content was accumulated in bone marrow, whereas hepatic LMW iron was not detectable after benzene intoxication to rats. Prior administration of single dose (250 micrograms/100 g body wt.) of Poly IC, an interferon inducer with immunomodulating potential was found to be ameliorate some of the adverse effects of benzene as well as restoration of hepatic architecture histologically. Superoxide dismutase activity, lipid peroxidation, total iron content and LMW iron content (bone marrow) were normalised. Pretreatment of animals with Poly IC was able to enhance the SRBC antibody titre in benzene-treated animals. This study suggests that the beneficial effects of Poly IC in the amelioration of the acute toxicity of benzene has clinical significance.

Pyridine prevents the clastogenicity of benzene but not of benzo[a]pyrene or cyclophosphamide.

Pyridine has been shown to be a much more potent inhibitor than other solvents of the metabolism and therefore the clastogenicity of benzene. In this report, pyridine prevented benzene-derived micronucleus formation in the bone marrow of ICR Swiss mice at much lower levels than xylene did. Time-course experiments did not indicate any delay in the peak micronucleus response to benzene caused by either pyridine or xylene. Similar experiments using pyridine with benzo[a]pyrene and pyridine with cyclophosphamide indicated that the effect of pyridine was specific for benzene. Benzo[a]pyrene (150 mg/kg) was inhibited by pyridine only at levels of 100 mg/kg or more, compared to inhibition of benzene (440 or 880 mg/kg) by pyridine at levels of 5 mg/kg. Cyclophosphamide was not inhibited at any level, and micronucleus formation was increased at lower ratios of pyridine to cyclophosphamide. These results provide indirect conformation of the work by others indicating that benzene is activated in part by a cytochrome P450 isozyme different from those activating benzo[a] pyrene or cyclophosphamide. Since DBA/2 mice (AHH-non-inducible) are more sensitive to benzene than C57Bl/6 mice (AHH-inducible), single and multiple treatments with benzene were compared by micronucleus response in these two strains. DBA mice were more responsive in all cases. Pretreatment with methylcholanthrene caused a greater response to benzene in DBA/2 mice, suggesting that the cytochrome P450 isozyme involved is one of the forms induced by methylcholanthrene independent of the high-affinity Ah receptor. It is hypothesized that more efficient activation of benzene by the unique cytochrome P450 isozyme, perhaps combined with relatively less conjugation, may result in a greater sensitivity of the bone marrow versus the liver, and of DBA/2 versus C57Bl/6 mice.

Suppression of bone marrow stromal cell function by benzene and hydroquinone is ameliorated by indomethacin.

Administration of benzene to mice will inhibit bone marrow stromal cell-supported hemopoiesis in culture. Hydroquinone, a major metabolite of benzene, will cause a similar inhibition of stromal cell function in vitro. Stromal cells produce both an inducer (colony-stimulating factor) and an inhibitor (prostaglandin E2; PGE2) of hemopoiesis. This research was conducted to determine if prostaglandin synthesis is involved in the suppression of stromal cell function by benzene and hydroquinone. Male B6C3F1 mice were administered benzene (100 mg/kg), indomethacin (1 mg/kg), or benzene plus indomethacin twice a day for 4 consecutive days. On Day 5 bone marrow cells were removed to determine the effect of treatment. In a second series of experiments mouse bone marrow stromal cells in culture were treated with hydroquinone (10(-7) to 10(-4) M), indomethacin (10(-6) M), or a combination of hydroquinone plus indomethacin. Stromal cell function was based on the ability of the treated stromal cells to support granulocyte/monocyte colony development in coculture. The results demonstrated that preadministration of indomethacin in vivo ameliorated benzene-induced inhibition of bone marrow stromal cell function. In vitro, indomethacin ameliorated hydroquinone toxicity to stromal cell function. Benzene administration in vivo induced elevated PGE2 in bone marrow samples which were prevented by preadministration of indomethacin. However, hydroquinone in vitro did not induce a consistent increase in PGE2 levels. These results suggested that toxicity to stromal cells was not due solely to increased prostaglandin synthetase activity.

Modulation of benzene toxicity by an interferon inducer (6MFA).

Repeated intraperitoneal administration of benzene (1.0 ml/kg body wt) for 3 days produced leucopenia, lymphocytopenia and an increased number of nucleated cells in the bone marrow and significantly decreased organ weights of thymus (P less than 0.001) and spleen (P less than 0.001) in female albino rats. Iron content, lipid peroxidation and superoxide dismutase activity of the liver and bone marrow were significantly increased as a result of benzene exposure. Prior administration of 6MFA, an interferon inducer with immunomodulating potential, was found to ameliorate some of the adverse effects of benzene as well as restoration of hepatic architecture histologically. Lipid peroxidation and iron content were both normalised, whereas superoxide dismutase activity was further increased and the number of lymphocytes and bone marrow cells returned to normal. Pretreatment of animals with 6MFA was able to enhance the SRBC antibody titre in benzene-treated immunosuppressed animals. The beneficial effects of 6MFA in the amelioration of the acute toxicity of benzene therefore assume certain significance.

An hypothesis for the induction of leukemia by benzene.

Benzene has the sad privilege of being the only industrial chemical inducing leukemia in susceptible individuals chronically exposed to its vapors. Hence, benzene has been included in the list of human carcinogens. Acute myeloblastic leukemia and erythroleukemia are typical examples of benzene leukemia. Most cases show some features in common: 1) development after many years of exposure and, in some cases many months after leaving the toxic atmosphere; 2) leucopenia or moderate degree of leucocytosis; and 3) splenohepatomegaly discrete or absent. Finding of an antecedent of pancytopenia reinforces the suspicion of benzene as the causative agent. There is still no agreement about the role played by benzene in chronic types of leukemia. In assessing diagnosis of benzene leukemia much importance has been attached by French authors and by myself to the demonstration of benzene in blood or in bone marrow aspirates or biopsies. Treatment of benzene hemopathy based on the oral administration of "anti-benzene compounds" such as methyl-donors and thiol-aminoacids is proposed here based on personal research in rabbits, in leukemic patients treated by benzene in the past and on myself as a volunteer. In pre-leukemic states, lowering the benzene burden of the bone marrow might prevent the further development of acute leukemia. Recently, I found out that: 1) benzene can be converted to phenol in the bone marrow independently of liver oxidizing enzymes; 2) benzene injected in the femoral artery of the rabbit can provoke histological changes at the isolated tibial marrow.

Modulation of benzene-induced lymphocytopenia in the rat by 2,4,5,2',4',5'-hexachlorobiphenyl and 3,4,3',4'-tetrachlorobiphenyl.

Repeated administration of benzene (440 mg/kg/day, s.c.) to 6-week-old male Fischer-344 rats resulted in a progressive decline in the number of circulating lymphocytes. Pretreatment of these animals with 2,4,5,2',4',5'-hexachlorobiphenyl (HCB) or 3,4,3',4'-tetrachlorobiphenyl (TCB) protected against benzene toxicity for as long as 7 days, but not after 10 days of repeated dosing. Representative phase I (mixed-function oxidase) and phase II (conjugating) enzyme activities were measured to determine whether the altered susceptibility to benzene toxicity in TCB- and HCB-pretreated rats could be correlated with changes in the profile of hepatic oxidative and detoxification pathways. Measurement of 7-ethoxycoumarin O-deethylase and benzphetamine N-demethylase activities indicated that the loss of protection by HCB or TCB against benzene toxicity after 7 days was not associated with changes in the activities of hepatic mixed-function oxidases inducible by 3-methylcholanthrene or phenobarbital. The time course for the stimulation by TCB and return to control values, of UDP-glucuronosyl transferase activity, a potential route for the elimination of benzene metabolites, mirrored the time course for the protection against toxicity. Epoxide hydratase activity was induced 2- to 3-fold by HCB. Although stimulation of this pathway could result in a decreased concentration of phenol, this activity did not decline with the loss of protection. Hepatic 10 000 X g supernatant fractions, prepared from livers of rats given TCB, were incubated with a non-saturating concentration of [14C] benzene (equivalent to 19 nmol/mg wet wt. tissue). Under these conditions the metabolism of benzene was depressed (40% of control) 2 days after pretreatment; after 14 days, the metabolism of benzene returned to control values. This pattern correlated temporarily with the protection against lymphocytopenia. The data indicate that the protection against benzene toxicity in rats pretreated with HCB or TCB is not necessarily related to the capacity of these compounds to induce phase I activities. In rats pretreated with TCB, the data suggest that decreasing the concentration of primary benzene metabolites, either by inhibiting the hepatic metabolism of benzene or increasing hepatic conjugation activity is an important factor modulating toxicity.

[Changes in lysosomal activity of beta-glucuronidase in leukocytes of rats exposed to benzene under selenic protection]. / Zmiany aktywnosci lizosomalnej beta-glukuronidazy w leukocytach szczurów przewlekle eksponowanych na benzen w oslonie selenowej.

The authors demonstrated that a chronic exposure to benzene caused in rats a reduction in the number of lymphocytes in peripheral blood, decrease in beta-glucoronidase (BG) activity in neutrophils and increase in BG-positive lymphocytes fraction. In addition, a failure was found in lysosomes of lymphocytes, revealed by scattering of a given enzyme in cytoplasma. Administration of 1.0 microgram/kg for 10 consecutive days before benzene exposure, did not prevent lymphocytopenia but hampered BG activity decrease in neutrophils as well as a damage to lymphocytic lysosomes. Administration of 5.0 microgram/kg for 10 successive days prior to benzene exposure, prevented lymphocytopenia and caused a reactive increase in the amount of neutrophils and resulted in an increase of BG-positive lymphocytes fraction, especially those with cytoplasmatic localization of enzyme. The results obtained demonstrate that only a smaller dose of selenium protected lysosomal membrane of lymphocytes from toxic effects of benzene, whereas both selenium doses protect BG in neutrophils.