Activation of carboxylic acids by pyrocarbonates. Synthesis of symmetric anhydrides and esters of N-protected amino acids using dialkyl pyrocarbonates as condensing reagents.

Activation of carboxylic acids was achieved via dialkyl pyrocarbonates (ROCO)2O, R = C2H5, i-C3H7, sec-C4H9, tert.-C4H9) in aprotic solvents in the presence tertiary amines. A convenient procedure for the preparation of carboxylic acid anhydrides from carboxylic acids and di-tert.-butyl pyrocarbonate in the presence of pyridine is reported. Analogously, di-isopropyl- or diethyl pyrocarbonate may be used in the presence of N-methylmorpholine (triethylamine). With pyridine, di-isopropyl- or diethyl pyrocarbonate carboxylic acids form isopropyl- or ethyl esters, respectively. A wide variety of esters were prepared in good yields in a one-pot procedure from carboxylic acids, including N-protected amino acids, and alcohols or from phenols by means of di-tert.-butyl pyrocarbonate in the presence of pyridine (Boc2O-pyridine system). t-Butyl esters of carboxylic acids were obtained by the same procedure with 4-dimethylaminopyridine. In the absence of carboxylic acid, with 4-dimethylaminopyridine Boc2O and alcohols generate alkyl tert.-butyl carbonates.     

Mutagenicity of 3-tert-butyl-4-hydroxyanisole (BHA) and its metabolites in short-term tests in vitro

The mutagenicity of 3-tert-butyl-4-hydroxyanisole (BHA) and its metabolites was investigated in the reverse mutation assay using S. typhimurium strains and the chromosomal aberration test in vitro using a Chinese hamster fibroblast cell line, CHL. BHA, tert-butylhydroquinone (BHQ), tert-butylquinone (BQ) and BHA dimer (diBHA) did not show any mutagenic potential with and without S9 mix in the reverse mutation assay. In addition to the above 4 chemicals, 3-tert-butyl-4,5-dihydroxyanisole (BHA-OH), 3-tert-butylanisole-4,5-quinone (BHA-o-Q), and tert-butylquinone oxide (BQO) were tested in the chromosomal aberration test. BHA, BHQ and BQ induced chromosomal aberrations only in the presence of S9 mix, while BHA-OH, BHA-o-Q and BQO induced chromosomal aberrations only without S9 mix. DiBHA, however, showed no clastogenic potential with and without S9 mix. The present findings suggest that BHA-OH, BHA-o-Q or BQO may contribute to the clastogenicity of BHA in the presence of S9 mix.     

Ceramide increases oxidative damage due to inhibition of catalase by caspase-3-dependent proteolysis in HL-60 cell apoptosis

We investigated through which mechanisms ceramide increased oxidative damage to induce leukemia HL-60 cell apoptosis. When 5 microm N-acetylsphingosine (C(2)-ceramide) or 20 microm H(2)O(2) alone induced little increase of reactive oxygen species (ROS) generation as judged by the 2'-7'-dichlorofluorescin diacetate method, 20 microm H(2)O(2) enhanced oxidative damage as judged by ROS accumulation, and thiobarbituric acid-reactive substance production after pretreatment with 5 microm C(2)-ceramide at least for 12 h. The treatment with a catalase inhibitor, 3-amino-1h-1,2,4-triazole, increased oxidative damage and apoptosis induced by H(2)O(2), and in contrast, purified catalase inhibited the enhancement of oxidative damage by H(2)O(2) in ceramide-pretreated cells, suggesting that the oxidative effect of ceramide is involved in catalase regulation. Indeed, C(2)-ceramide inhibited the activity of immunoprecipitated catalase and decreased the levels of catalase protein in a time-dependent manner. Moreover, acetyl-Asp-Met-Gln-Asp-aldehyde, which dominantly inhibited caspase-3 and blocked the increase of oxidative damage and apoptosis due to C(2)-ceramide-induced catalase depletion at protein and activity levels. In vitro, active and purified caspase-3, but not caspase-6, -8, and -9, inhibited catalase activity and induced the proteolysis of catalase protein whereas these in vitro effects of caspase-3 were blocked by acetyl-Asp-Met-Gln-Asp-aldehyde. Taken together, it is suggested that H(2)O(2) enhances apoptosis in ceramide-pretreated cells, because ceramide increases oxidative damage by inhibition of ROS scavenging ability through caspase-3-dependent proteolysis of catalase.     

Participation of active oxygen species in the induction of chromosomal aberrations by cadmium chloride in cultured Chinese hamster cells

The effect of various scavengers of active oxygen species on the induction of chromosomal aberrations by cadmium chloride (CdCl2) was investigated in cultured Chinese hamster V79 cells. Incidences of chromosomal aberrations by CdCl2 were partially or fully reduced by the presence of catalase, mannitol (a scavenger of hydroxyl radicals) and butylated hydroxytoluene (BHT, an antioxidant). These findings may indicate participation of the active oxygen species such as hydrogen peroxide (H2O2) or hydroxyl radicals in the clastogenicity of cadmium. In contrast, superoxide dismutase (SOD) and dimethylfuran (a scavenger of singlet oxygen) did not influence incidences of chromosomal aberrations by CdCl2. These results suggest that superoxide anion and singlet oxygen are not directly involved in the clastogenicity of the metal. The presence of aminotriazole (an inhibitor of catalase) increased incidences of chromosomal aberrations by CdCl2. This emphasizes participation of H2O2 in the clastogenicity of cadmium.     

Are free radicals involved in tumor promotion?

Previously it was shown that lipophilic analogs of a free-radical scavenger, 2(3)-tert-butyl-4-hydroxyanisole (BHA), inhibit ornithine decarboxylase (ODC) activity which is induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in mouse epidermis. With regard to this antitumor-promoting effect, eight analogs of BHA (2- and 3-BHA, 2-t-butyl-1, 4-dimethoxybenzene methyl-BHA), t-butylhydroquinone (t-BHQ), p-hydroquinone (HQ), 4-hydroxyanisole, phenol and 2-t-butylphenol) are evaluated herein for their antioxidant capacities for scavenging superoxide anions (O-2), of inhibiting lipid peroxidation and of inhibiting chemiluminescence (CL) in TPA-activated polymorphonuclear leukocytes (PMNs), an event associated with oxy-radical production. None of the analogs reacted with O-2, while 2- and 3-BHA suppressed the formation of O-2 by TPA-activated PMNs. T-BHQ underwent autoxidation in aqueous solution, reducing molecular oxygen and increasing the levels of O-2 that were formed chemically, enzymatically and cellularly. However, all of the phenolic antioxidant analogs of BHA inhibited TPA-stimulated CL in PMNs and ascorbate-initiated lipid peroxidation, while methyl-BHA (a non-antioxidant analog) was inactive. The inhibitory activities of these analogs for lipid peroxidation were related to both their lipophilic and antioxidant properties and corresponded favorably with their inhibitory activities for TPA-induced ODC activities in mouse epidermis. On the other hand, inhibition of the CL response by these antioxidants was independent of their lipophilicity and compared less favorably with their capacities to antagonize phorbol ester-induced ODC activity. These results imply that lipophilic BHA analogs inhibit TPA-induced ODC activity by scavenging free radicals other than O-2. Furthermore, the fact that t-BHQ was the most potent inhibitor of CL, lipid peroxidation and ODC activity and simultaneously reduced molecular oxygen, suggests the possibility that O-2 may act as a precursor to the formation of free radicals which are reactive with t-BHQ and more directly involved in the process of tumor promotion.     

Autoxidation of extracellular hydroquinones is a causative event for the cytotoxicity of menadione and DMNQ in A549-S cells

Cytotoxicity of 1,4-naphthoquinones has been attributed to intracellular reactive oxygen species (ROS) generation through one-electron-reductase-mediated redox cycling and to arylation of cellular nucleophiles. Here, however, we report that in a subclone of lung epithelial A549 cells (A549-S previously called A549-G4S (Watanabe, et al., Am. J. Physiol. 283 (2002) L726-736), the mechanism of ROS generation by menadione and by 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), and therefore that of cytotoxicity, differs from the paradigm. Ninety percent of H(2)O(2) generation by both the quinones can be prevented by dicumarol, an inhibitor of NAD(P)H quinone oxidoreductase (NQO1), at the submicromolar level, regardless of the quinone concentrations. Exogenous SOD also inhibits H(2)O(2) production at low but not high concentrations of the quinones, especially DMNQ. Thus, at low quinone concentrations, superoxide-driven hydroquinone autoxidation accounts for more than half of H(2)O(2) generation by both quinones, whereas at high quinone concentrations, especially for DMNQ, comproportionation-driven hydroquinone autoxidation becomes the predominant mechanism. Hydroquinone autoxidation appears to occur predominantly in the extracellular environment than in the cytosol as extracellular catalase can dramatically attenuate quinone-induced cytotoxicity throughout the range of quinone concentrations, whereas complete inactivation of endogenous catalase or complete depletion of intracellular glutathione has only a marginal effect on their cytotoxicity. Finally, we show evidence that ROS production is a consequence of the compensatory defensive role of NQO1 against quinone arylation.     

Hydrogen peroxide downregulates human organic anion transporters in the basolateral membrane of the proximal tubule

Hydrogen peroxide (H2O2) is known to be involved in drug-induced and ischemic proximal tubular damage. The purpose of this study was to elucidate the effects of hydrogen peroxide on organic anion transport mediated by human organic anion transporters 1 and 3 (hOAT1 and hOAT3), which are localized at the basolateral side of the proximal tubule. For this purpose, we established and utilized the second segment of the proximal tubule cells from mice stably expressing hOAT1 or hOAT3 (S2 hOAT1 or S2hOAT3, respectively). H2O2 induced a dose- and a time-dependent decrease in organic anion transport mediated by hOAT1 and hOAT3. Kinetic analysis revealed that H2O2 decreased the Vmax, but not Km of organic anion transport both in S2hOAT1 and S2hOAT3. The effects of gentamicin, known to induce proximal tubular damage via the production of H2O2, on the organic anion transporters were also examined. Gentamicin induced a significant decrease in organic anion transport in S2hOAT1 but not S2hOAT3. H2O2-induced decrease in organic anion transport was significantly inhibited by pretreatment with pyruvate as well as catalase, whereas the gentamicin-induced decrease was significantly inhibited by pretreatment with pyruvate but not with catalase. In conclusion, these results suggest that H2O2, which is produced during tubular injuries, downregulates organic anion transport mediated by both hOAT1 and hOAT3, leading to further modulation of pathophysiology.     

Oxidants,antioxidants and carcinogenesis

Reactive oxygen metabolites (ROMs), such as superoxide anions (O2*-) hydrogen peroxide (H2O2), and hydroxyl radical (*OH), malondialdehyde (MDA) and nitric oxide (NO) are directly or indirectly involved in multistage process of carcinogenesis. They are mainly involved in DNA damage leading sometimes to mutations in tumour suppressor genes. They also act as initiator and/or promotor in carcinogenesis. Some of them are mutagenic in mammalian systems. O2*-, H2O2 and *OH are reported to be involved in higher frequencies of sister chromatid exchanges (SCEs) and chromosome breaks and gaps (CBGs). MDA, a bi-product of lipid peroxidation (LPO), is said to be involved in DNA adduct formations, which are believed to be responsible for carcinogenesis. NO, on the other hand, plays a duel role in cancer. At high concentration it kills tumour cells, but at low concentration it promotes tumour growth and metastasis. It causes DNA single and double strand breaks. The metabolites of NO such as peroxynitrite (OONO-) is a potent mutagen that can induce transversion mutations. NO can stimulate O2*-/H2O2/*OH-induced LPO. These deleterious actions of oxidants can be countered by antioxidant defence system in humans. There are first line defense antioxidants such as superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT). SOD converts O2*- to H2O2, which is further converted to H2O with the help of GPx and CAT. SOD inhibits *OH production. SOD also act as antipoliferative agent, anticarcinogens, and inhibitor at initiation and promotion/transformation stage in carcinogenesis. GPx is another antioxidative enzyme which catalyses to convert H2O2, to H2O. The most potent enzyme is CAT. GPx and CAT are important in the inactivation of many environmental mutagens. CAT is also found to reduce the SCE levels and chromosomal aberrations. Antioxidative vitamins such as vitamin A, E, and C have a number of biological activities such as immune stimulation, inhibition of nitrosamine formation and an alteration of metabolic activations of carcinogens. They can prevent genetic changes by inhibiting DNA damage induced by the ROMs. Therefore, these antioxidants may be helpful in the treatment of human cancer. However, detailed studies are required to draw a definite conclusion.     

TNFalpha induces chromosomal abnormalities independent of ROS through IKK, JNK, p38 and caspase pathways

A role for pro-inflammatory cytokines in inflammation-related cancers has been suggested, but mechanisms are not defined. Here, we demonstrate that treatment of HeLa cells with TNFalpha increases chromosomal aberration. In contrast, IL-1beta did not increase, but rather decreased chromosomal aberration. TNFalpha and IL-1beta increased the production of H2O2 to similar levels in cells, suggesting that increased production of reactive oxygen species might not be the premier factor involved. Reducing H2O2 through overexpression of catalase or treatment of cells with NAC or BHA did not have an effect on TNF-induced chromosomal aberration. TNFalpha-induced NO production has been implicated in DNA damage. Inhibiting NO did not reduce TNF-induced chromosomal aberration. Inhibiting IKK, JNK, and p38 kinase as well as caspases decreased TNF-induced chromosomal aberration, and a correlation between TNF-induced apoptosis and CA generation was not found. Single-strand DNA breaks give rise to double-strand breaks, which then results in chromosomal breaks, when replication forks reach the single-strand breaks during S-phase. In cells progressing through S-phase, TNFalpha activation of IKK, JNK, and p38 is significantly reduced. However, these kinases were activated by IL-1beta in S-phase. The possibility that these pathways, in a TNF-specific manner, may regulate either the generation of single- and double-strand breaks or their repair, thereby resulting in increased chromosomal aberration, is discussed.     

Induction of chromosomal aberrations in active oxygen-generating systems. I. Effects of paraquat in Chinese hamster cells in culture

A possible role for the superoxide anion radical (O2-) in the clastogenicity of paraquat (PQ) was investigated in cultured Chinese hamster cells. When cells were treated with 0.8 mg/ml of PQ for 3 h followed by 21 h of recovery time, structural chromosome aberrations were induced in about 50% of the metaphases examined. Almost all aberrations were of the chromatid-type and involved exclusively gaps and breaks. The induction of chromosomal aberrations by PQ was enhanced by a 1-h pretreatment with diethyldithiocarbamate, an inhibitor of superoxide dismutase. Diethyl maleate, a glutathione scavenger, also enhanced the induction of chromosomal aberrations, but 3-aminotriazole, an inhibitor of catalase, showed no such effects. Enhanced induction of chromosomal aberrations was also observed when PQ-treated cells were cultured at a high oxygen concentration (80%). The present results suggest that the production of chromosomal aberrations by PQ may be directly or indirectly related to the generation of O2-, but not to the formation of hydrogen peroxide by the dismutation reaction of O2- or of other active oxygen species including the hydroxyl radical and singlet oxygen.     

Influence of salicylic acid on H2O2 production, oxidative stress, and H2O2-metabolizing enzymes. Salicylic acid-mediated oxidative damage requires H2O2.

We investigated how salicylic acid (SA) enhances H2O2 and the relative significance of SA-enhanced H2O2 in Arabidopsis thaliana. SA treatments enhanced H2O2 production, lipid peroxidation, and oxidative damage to proteins, and resulted in the formation of chlorophyll and carotene isomers. SA-enhanced H2O2 levels were related to increased activities of Cu,Zn-superoxide dismutase and were independent of changes in catalase and ascorbate peroxidase activities. Prolonging SA treatments inactivated catalase and ascorbate peroxidase and resulted in phytotoxic symptoms, suggesting that inactivation of H2O2-degrading enzymes serves as an indicator of hypersensitive cell death. Treatment of leaves with H2O2 alone failed to invoke SA-mediated events. Although leaves treated with H2O2 accumulated in vivo H2O2 by 2-fold compared with leaves treated with SA, the damage to membranes and proteins was significantly less, indicating that SA can cause greater damage than H2O2. However, pretreatment of leaves with dimethylthiourea, a trap for H2O2, reduced SA-induced lipid peroxidation, indicating that SA requires H2O2 to initiate oxidative damage. The relative significance of the interaction among SA, H2O2, and H2O2-metabolizing enzymes with oxidative damage and cell death is discussed.     

Dopamine Oxidation Alters Mitochondrial Respiration and Induces Permeability Transition in Brain Mitochondria

Both reactive dopamine metabolites and mitochondrial dysfunction have been implicated in the neurodegeneration of Parkinson's disease. Dopamine metabolites, dopamine quinone and reactive oxygen species, can directly alter protein function by oxidative modifications, and several mitochondrial proteins may be targets of this oxidative damage. In this study, we examined, using isolated brain mitochondria, whether dopamine oxidation products alter mitochondrial function. We found that exposure to dopamine quinone caused a large increase in mitochondrial resting state 4 respiration. This effect was prevented by GSH but not superoxide dismutase and catalase. In contrast, exposure to dopamine and monoamine oxidase-generated hydrogen peroxide resulted in a decrease in active state 3 respiration. This inhibition was prevented by both pargyline and catalase. We also examined the effects of dopamine oxidation products on the opening of the mitochondrial permeability transition pore, which has been implicated in neuronal cell death. Dopamine oxidation to dopamine quinone caused a significant increase in swelling of brain and liver mitochondria. This was inhibited by both the pore inhibitor cyclosporin A and GSH, suggesting that swelling was due to pore opening and related to dopamine quinone formation. In contrast, dopamine and endogenous monoamine oxidase had no effect on mitochondrial swelling. These findings suggest that mitochondrial dysfunction induced by products of dopamine oxidation may be involved in neurodegenerative conditions such as Parkinson's disease and methamphetamine-induced neurotoxicity.     

Inhibition of Staphylococcus aureus by H2O2-producing Lactobacillus gasseri isolated from the vaginal tract of cattle

Infectious diseases are the major reproductive complication during postpartum. The bacteria that colonize the postpartum uterus of cattle are specific as well as opportunistic pathogens that include Staphylococcus aureus. The antibiotics and antiseptic agents used in the treatment of postpartum acute infections induce residues in foods, spread of bacterial resistance, increase in financial costs and failures in defense mechanisms of the host. Preventive treatment with probiotic products could decrease the use of antibiotics in dairy farming systems. Lactobacilli are present in the vaginal microflora of healthy cows. They can prevent pathogen colonization by mechanisms such as the production of antagonistic substances as lactic acid, H2O2, or bacteriocins. The aim of the present study was the selection of H2O2 generating lactobacilli from a group of 72 strains isolated from the vagina of cattle. Both Lactobacillus gasseri CRL1421 and Lactobacillus gasseri CRL1412, which share some probiotic properties, produce H2O2, detected by the plate colorimetric method. They were chosen to study the kinetics of H2O2 production under different culture conditions. Both microorganisms produced greater amounts of H2O2 in aerated than in static cultures. As L. gasseri CRL1421 had a greater capacity to generate H2O2, associative cultures with this strain and S. aureus were conducted. A significant decrease in the growth of the pathogen was detected after culture for 6h, this effect being greater under aerated conditions. The addition of catalase to mixed cultures partially abolished the inhibition, an effect that could be attributed to the combined action of H2O2 and other antagonistic metabolites. The simultaneous addition of catalase and NaOH to these cultures restored S. aureus growth. This observation suggests that the inhibition was produced by the combination of H2O2 and lactic acid, both released by the lactobacilli. Electron microphotographs showed the damage caused by the lactobacilli supernatant on the pathogenic cells. The treatment of S. aureus with lactic acid and hydrogen peroxide evidenced that each metabolite produced a different type of morphological damage. The number of viable cells obtained agrees with the electron microscopy observations. The results support the idea that L. gasseri CRL1421 could be successfully included in a probiotic product to prevent S. aureus infection in cows.     

In vitro stimulation of microsomal diethyl nitrosamine deethylase activity by vitamin K3 (menadione).

Vitamin K3 (menadione) has been found to stimulate diethyl nitrosamine (DEN)-deethylase activity in rat liver microsomes. The vitamin also takes care of the inhibitory effect of the anaerobic conditions as well as those of cytochrome poisons like sodium azide and sodium cyanide, possibly through production of active oxygen species. The enzyme was also stimulated by H2O2 and SOD and inhibited by catalase, thereby suggesting that H2O2 or some derivatives of it may be the active oxygen species involved in the reaction.     

Activated oxygen generation by a primaquine metabolite: inhibition by antioxidants derived from Chinese herbal remedies.

Primaquine is an important antimalarial drug which causes hemolytic anemia in patients with glucose-6-phosphate dehydrogenase (G6PDH) deficiency, probably due to oxidant generation by its metabolites. One of primaquine's metabolites, 5,6-dihydroxy-8-aminoquinoline (AQD), was found to cause chemiluminescence (CL) in vitro when incubated in the presence of luminol. This CL is inhibited by catalase and deferoxamine, unaffected by mannitol, and stimulated by superoxide dismutase (SOD), suggesting that it is mediated by H2O2. Three antioxidants (daphnetin, ferulate, and maltol), derived from Chinese herbal remedies, inhibited AQD- and H2O2-mediated CL, whereas a fourth, anisodamine, had no effect. Daphnetin also potently inhibited H2O2-mediated lipid peroxidation as measured by the production of thibarbituric acid reacting substances (TBARS). Thus, the possibility is raised that an antioxidant might be able to mitigate the oxidant hemolytic effects of primaquine.     

Copper redox-dependent activation of 2-tert-butyl(1,4)hydroquinone: formation of reactive oxygen species and induction of oxidative DNA damage in isolated DNA and cultured rat hepatocytes

The biotransformation of butylated hydroxyanisole (BHA), a possible carcinogenic food antioxidant, includes o-demethylation to 2-tert-butyl(1,4)hydroquinone (TBHQ) which can subsequently be oxidized to 2-tert-butyl(1,4)paraquinone (TBQ). In this study, we have examined the capacity of Cu, a nuclei- and DNA-associated transition metal, to mediate the oxidation of TBHQ. In phosphate buffered saline (PBS), autooxidation of TBHQ to TBQ was not detectable, while Cu(II) at micromolar concentrations strongly catalyzed the oxidation of TBHQ to TBQ. Oxidation of TBHQ by Cu(II) was accompanied by the utilization of O(2) and the concomitant generation of H(2)O(2). Using electron spin resonance spectroscopy, it was observed that Cu(II) mediated the one electron oxidation of TBHQ to a semiquinone anion radical. The formation of a semiquinone anion radical, the utilization of O(2) and the generation of H(2)O(2) and TBQ could be completely blocked by bathocuproinedisulfonic acid (BCS) and reduced glutathione (GSH), two Cu(I)-chelators. 4-Pyridyl-1-oxide-N-tert-butylnitrone (POBN)-spin trapping experiments showed that the reaction of TBHQ with Cu(II) resulted in the generation of POBN-CH(3) and POBN-CH(OH)CH(3) adducts in the presence of dimethyl sulfoxide (DMSO) and ethanol, respectively, suggesting the formation of hydroxyl radical or a similar reactive intermediate. The formation of POBN-CH(3) adduct from the TBHQ/Cu(II)+DMSO could be completely inhibited by catalase, GSH or BCS, indicating that the hydroxyl radical or its equivalent is generated from the interaction of H(2)O(2) with Cu(I). Incubation of supercoiled phiX-174 plasmid DNA with the TBHQ/Cu(II) resulted in extensive DNA strand breaks, which could be prevented by catalase or BCS. Incubation of rat hepatocytes with TBHQ in PBS led to increased formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in nuclear DNA. The TBHQ-induced formation of 8-OHdG was markedly reduced in the presence of cell permeable Cu(I)-specific chelator, bathocuproine or neocuproine, suggesting that a Cu(II)/Cu(I) redox mechanism may also be involved in the induction of oxidative DNA damage by TBHQ in hepatocytes. Taken together, the above results conclusively demonstrate that the activation of TBHQ by Cu(II) results in the formation of TBQ, semiquinone anion radical and reactive oxygen species (ROS), and that the ROS formed may participate in oxidative DNA damage in both isolated DNA and intact cells. These reactions may contribute to the carcinogenicity as well as other biochemical activities observed with BHA in animals. To our knowledge this study provides the first evidence that endogenous cellular Cu may be capable of bioactivating TBHQ, leading to oxidative DNA damage in cultured cells.     

Chromosome analysis of human spermatozoa following in vitro exposure to cyclophosphamide, benzo(a)pyrene and N-nitrosodimethylamine in the presence of rat liver S9

For the purpose of assessing mutagenic effects (clastogenicity) of metabolites derived from chemical mutagens/carcinogens on human sperm chromosomes, spermatozoa were exposed in vitro to cyclophosphamide (CP), benzo(a)pyrene (BP) or N-nitrosodimethylamine (NDMA) for 2h in the presence or absence of rat liver S9, a metabolic activator of these chemicals. After in vitro fertilization between human spermatozoa and zona-free hamster oocytes, chromosome complements of sperm origin were analyzed cytogenetically.In the absence of S9, none of three chemicals (20 microg/ml CP, 200 microg/ml BP and 20mg/ml NDMA) caused a significant increase in spermatozoa with structural chromosome aberrations (8.6, 10.0 and 7.5%), as compared with their matched controls (10.9, 11.0 and 8.5%). In the presence of S9, however, a significant increase in chromosomally abnormal spermatozoa was observed in CP (37.1%, P < 0.001) and BP (31.0%, P < 0.001), indicating that enzymatic activation of CP and BP induced chromosomal abnormalities in human sperm. In contrast, NDMA did not induce chromosome aberrations in human spermatozoa by S9 treatment, although positive results have been observed in somatic cells. The present results on in vitro clastogenicity of CP, BP and NDMA are consistent with the results in previous in vivo studies with murine spermatozoa. Our S9/human sperm chromosome assay seems to be useful for estimation of hereditary risk of chemicals in human. Because most chemicals need metabolic activation to bind to DNA.     

Different cytotoxic and clastogenic effects of epigallocatechin gallate in various cell-culture media due to variable rates of its oxidation in the culture medium

Positive genotoxicity results are often observed using mammalian cells in culture with agents that are not in vivo genotoxins. We here illustrate one possible explanation: interaction of test chemicals with the cell-culture media used. We find that the toxicity and clastogenicity of epigallocatechin gallate (EGCG) to Chinese Hamster ovary (CHO) cells is affected by the culture medium used and appears largely or entirely due to variable rates of formation of hydrogen peroxide (H(2)O(2)) by chemical reactions of EGCG with the culture media. Catalase decreased EGCG toxicity substantially. Of seven different types of commonly used media evaluated, F-10 and F-12 nutrient mixtures were the least prone to produce this artefact. Although it generated H(2)O(2) in the culture media, ascorbate was not toxic to CHO cells because the H(2)O(2) levels achieved were insufficient to kill these cells. Thus, the culture medium, the cell type and the presence or absence of catalase (e.g. its variable amounts in S9 fractions) must be taken into account in in vitro genotoxicity testing.     

Induction of oxidative DNA damage in anaerobes

We compared oxidative DNA damage in strictly anaerobic Prevotella melaninogenica, aerotolerant anaerobic Bacteroides fragilis, and facultative anaerobic Salmonella typhimurium after exposure to O2 or H2O2. Using HPLC with electrochemical detection, we measured 8-hydroxydeoxyguanosine (8OHdG) as a damage marker. O2 induced 8OHdG in P. melaninogenica but not in B. fragilis, which shows catalase activity, or in S. typhimurium. In P. melaninogenica, with catalase, O2 induced less 8OHdG; superoxide dismutase had no effect; with glucose and glucose oxidase, O2 induced more 8OHdG. H2O2 also markedly increased 8OHdG. O2 was suggested to induce 8OHdG through H2O2. O2 or H2O2 decreased survival only in P. melaninogenica. Highly sensitive to oxidative stress, P. melaninogenica could prove useful for investigating oxidative DNA damage.