Rat Liver Microsomal System Research Articles

Inhibitory activity of epigallocatechin gallate (EGCg) in paraquat-induced microsomal lipid peroxidation—a mechanism of protective effects of EGCg against paraquat toxicity

Recently we have reported that epigallocatechin gallate (EGCg), a major component of Japanese green tea, significantly increased the survival rate of paraquat (Pq) poisoned mice. This paper describes two biochemical activities of EGCg, which relate to its protective effects against Pq toxicity. EGCg inhibited Pq-induced microsomal malondialdehyde (MDA) productions in rat liver microsome system containing 40 μM FeSO 4. Forty micromolar EGCg inhibited MDA production significantly. EGCg may inhibit the Pq-induced MDA production by at least two mechanisms. One may be iron-chelating activity as the inhibition disappeared when excess amounts of FeSO 4 were added to the reaction mixture, which indicated that EGCg reduced iron driven lipid peroxidation by pulling out available irons in the reaction mixture. The other is radical scavenging activity. EGCg scavenged DMPO-OOH spin adducts generated by the microsome–Pq system. The dose response curve of EGCg was similar to that obtained by ascorbic acid which is a typical water-soluble radical scavenger. Although ascorbic acid had a potential activity of scavenging superoxide radicals, it can not be recommended to use for the treatment of Pq poisoning, because ascorbic acid acts as a pro-oxidant in the presence of free transition metal ions by accelerating the Fenton reaction (Fe 2++H 2O 2→Fe 3++OH −+OH ), which is responsible for lipid peroxidation. On the contrary, EGCg inhibited iron-driven lipid peroxidation presumably not only by chelating to Fe ions but also by scavenging superoxide radicals, which are responsible for the reduction of ferric (Fe 3+) to ferrous (Fe 2+) that catalyzes the Fenton reaction. Chelating and radical scavenging activity of EGCg can be expected simultaneously in the occurrence of Pq toxicity, which may explain the protective effects of EGCg against Pq toxicity.

Differential anti-lipid peroxidative activity of melatonin

Scavenging activities of melatonin, which is a pineal secretory product and functions in circadian biology, and its related compounds against reactive oxygen species such as superoxide anion radical, hydrogen peroxide, hydroxyl radical and singlet oxygen as well as organic peroxide radical (t-BuOO*) were evaluated chemically by using electron spin resonance-spin trap and chemiluminescence methods. Antioxidative activity of the compounds was estimated by IC50 value (microM), 50% inhibiting concentration of a compound against reactive oxygen species formed in each system, and the second-order rate constants (k2) for the reactions of the compounds and superoxide anion radical or hydroxyl radical. Because melatonin has exhibited the highest scavenging activity against t-BuOO*, the biochemical anti-lipid peroxide radical scavenging activities of melatonin were examined. We found that melatonin exhibits higher anti-lipid peroxidative activity in the rat brain microsomes than in the rat liver microsomal and liposomal systems, suggesting that melatonin may function as a treatment for reactive oxygen species-related diseases of the brain.

Free radical scavenging action of medicinal herbs from Mongolia.

In the present study we evaluated the free radical scavenging action of some medicinal herbs growing in Mongolia. The aqueous extract of nine herbs Chamenerion angustifolium (Ch.ang), Equisetum arvense (Eq.arv), Gentiana decumbens (Gn.dec), Geranium pratense (Gr.pra), Lomatogonium carinthiacum (L.car), Nonea poulla (N.pl), Phodococcum vitis-idaea (Ph.v), Sphallerocarpus gracilis (Sph.gr), Stellera chamaejasme (St.cha) were used in the present experiment. The free radical scavenging action was determined in vitro and ex vivo by using electron spin resonance (ESR) spectrometer and chemiluminescence (CL) analyzer. The results showed that extracts of Ch.ang, Gn.dec, Gr.pra, L.car, N.pl, Ph.v, Sph.gr and St. cha possess strong scavenging action of 1,1-diphenyl-2-picrylhydrazyl, superoxide and hydroxyl radicals. On the other hand, the radical scavenging action of Eq.arv was low. Extracts of N.pl and Ph.v markedly inhibited the CL generated from rat liver microsomal cytochrome P450 system whereas the CL was moderately inhibited by Eq.arv, Gn.dec, Gr.pra, L.car and St.cha. The extracts of Ch.ang and Sph.gr did not decrease the CL generation. Ch.ang, Gr.pra, L.car, N.pl, Ph.v and St.cha also depressed reactive oxygen production from polymorphonuclear leukocytes stimulated by phorbol-12-myristate acetate ex vivo. Thus it was confirmed that the medicinal herbs from Mongolia possess high antioxidant potency in vitro and ex vivo.

Mitochondrial permeability transition induced by 1-hydroxyethyl radical

Impairment of mitochondrial functions has been found in ethanol-induced liver injury. Ethanol can be oxidized to the 1-hydroxyethyl radical (HER) by rat liver microsomal systems. Experiments were carried out to evaluate the ability of HER to cause mitochondrial swelling as an indicator of the mitochondrial permeability transition (MPT). Electron spin resonance (ESR) spectroscopy was used to detect HER and to study its interaction with mitochondria. The ESR signal intensity of the spin adduct formed from α-(4-pyridyl-1-oxide) N- tert-butylnitrone (POBN) and HER generated from either a thermic decomposition of 1,1′-dihydroxyazoethane (DHAE) or a Fenton reaction system containing ethanol was markedly diminished by the addition of mitochondria, indicating an interaction between HER and mitochondria. Exposure of rat liver mitochondria to HER generated from either system caused swelling, as reflected by a decrease in absorbance at 540 nm, in a HER concentration-dependent and a cyclosporin A–sensitive manner. Mitochondrial swelling was also induced in the Fenton reaction system without ethanol. The DHAE-dependent generation of HER in mitochondrial suspension resulted in a decrease of membrane protein thiols and collapse of the membrane potential (Δ Ψ). The swelling induced by HER was prevented by glutathione and vitamin E, but not by superoxide dismutase. Catalase did not prevent the swelling caused by the acetaldehyde/hydroxylamine O-sulfonate (HOS) system, but was inhibitory in the Fenton reaction system with or without ethanol. These results indicate that HER, as well as hydroxyl radical, can induce the MPT, and suggest the possibility that the collapse of Δ Ψcaused by HER may, at least in part, contribute to impairment of mitochondrial function caused by ethanol and in ethanol-induced liver injury.

Some sulfur-containing metabolites of tri-n-butyltin chloride in male rats.

In an attempt to elucidate metabolic destination of TBTO, sulfur-containing metabolites were investigated in the urine. Tri-n-butyltin chloride (TBTC), tri-n-butyltin oxide (TBTO), and their in vitro metabolites in rat liver microsomal enzyme systems, di-n-butyl(3-hydroxybutyl)tin chloride (T3OH), di-n-butyl(3-oxobutyl)tin chloride (T3CO), dibutyltin dichloride (DBTC), and monobutyltin trichloride (MBTC), were intraperitoneally administered to rats. In particular, administration of T3OH and T3CO gave higher amounts of mercapturic acid derivatives, such as N-acetyl-S-(3-oxobutyl)-L-cysteine (3CO-MA) and N-acetyl-S-(3-hydroxybutyl)-L-cysteine (3OH-MA), than TBTC or TBTO. On the other hand, DBTC and MBTC did not yield measurable amounts of 3CO-MA and/or 3OH-MA. The appearance of organotin metabolites in urine indicates that T3OH, T3CO, and hypothesized secondary metabolites, such as n-butyl(3-hydroxybutyl)(3-oxobutyl)tin chloride, n-butyl(3-hydroxybutyl)(4-hydroxybutyl)tin chloride, etc., are subject to the action of glutathione S-transferase to give mercapturic acid derivatives. These sulfur-containing metabolites (3CO-MA and 3OH-MA) were also found in control rat urine.

A Novel Bioactivation Mechanism of Phosphoramidate Insecticides

In order to elucidate the activation mechanism of phosphoramidate insecticides, 2,4-dichlorophenyl methylN-alkylphosphoramidates (alkyl: methyl, ethyl, propyl, isoproppyl, and butyl) were chosen as model compounds and compared with isofenphos oxon. Although they showed a potent insecticidal activity against susceptible housefly except for butyl homologue, theirin vitroacetylcholinesterase (AChE) inhibitory activity was very poor.N-Desalkyl compound has been known as an activated form. When bovine erythrocyte AChE inhibition ofN-isopropyl homologue and isofenphos oxon were carried out in rat liver microsomal system, they gave a strong inhibitory activity under the oxidative condition with NADPH, but, in the presence of SKF-525A or piperonyl butoxide as a mixed function oxidase (MFO) inhibitor, isofenphos oxon lost all the inhibitory activity. On the other hand,N-isopropyl homologue partly retained its AChE activity and completely lost the activity by the addition of methimazole as a flavin-containing monooxygenase (FMO) inhibitor. Comparison of these data with insecticidal activity suggested the involvement of another bioactivation mechanism. Peracid oxidation as a model of biological oxidation resulted in the production of des-N-alkyl compound (HO-PO) in addition toN-desalkyl compound (H2N-PO), which suggested the structure of the activated form as an oxidized form of phosphoramide moiety of the starting compound. Three structures were considered from a computational chemistry point of view.N-Oxide structure (RNH(O)-PO) is isomerized toO-aminophosphate structure (RNHO-PO) depending on the alkyl substituent on the nitrogen atom, and the latter is the intermediate to give des-N-alkyl compound, which has no AChE inhibitory action. The formation ofO-aminophosphate structure from the adduct of the starting compound with peracid was analyzed using model compounds by the theory of Frontier orbital. Also,O-aminophosphate structure might be producedviaphosphorus oxyoxonate structure (RNH-P(OO)). The possibility of hydroxylamine structure (RN(OH)-PO) to giveN-oxide orO-aminophosphate structure was small if any in this model phosphoramidate compounds. In any way,O-aminophosphate structure is most probable as a novel activated form mainly formed by FMO or peracid.

Antimutagenicity of sweetpotato (Ipomoea batatas) roots.

Antimutagenicity of the water extracts prepared from the storage roots of four varieties of sweetpotato with different flesh colors was investigated using Salmonella typhimurium TA 98. The extract from the whole roots of the purple-colored Ayamurasaki variety effectively decreased the reverse mutation induced not only by Trp-P-1, Trp-P-2, IQ, B[a]P, and 4-NQO but also by dimethyl sulfoxide extracts of grilled beef. Comparison of the inhibitory activity of the extracts from the normal Ayamurasaki and its anthocyanin-deficient mutant one suggested that the anthocyanin pigment in the flesh decreases the mutagenic activity of the mutagens as heterocyclic amines. Two anthocyanin pigments purified from purple-colored sweet-potato, 3-(6,6'-caffeylferulylsophoroside)-5-glucoside of cyanidin (YGM-3) and peonidin (YGM-6) effectively inhibited the reverse mutation induced by heterocyclic amines, Trp-P-1, Trp-P-2, and IQ in the presence of rat liver microsomal activation systems.

A labile sulfur in trisulfide affects cytochrome P-450 dependent lipid peroxidation in rat liver microsomes

The effects of trisulfide derivatives were studied on cytochrome P-450-dependent lipid peroxidation using rat liver microsomal systems. Cytochrome P-450-dependent lipid peroxidation was induced by carbon tetrachloride or tert-butylhydroperoxide and was evident by an increase in thiobarbituric acid-reactive substances (TBA-RS) and oxygen consumption. In these cytochrome P-450-dependent lipid peroxidation systems, pretreatment of microsome with trisulfide derivatives (cystine trisulfide and thiocyclam) significantly inhibited TBA-RS formation and oxygen consumption compared with disulfide or thiol analogs (cystine, nereistoxin, or cysteine). The labile sulfur contained in trisulfide disappeared during incubation with liver microsomes. In the CCl 4-induced lipid peroxidation system, the cytochrome P-450 level and NAD(P)H-cytochrome P-450 reductase activity were significantly decreased by the addition of trisulfide derivatives. Therefore, in cytochrome P-450-dependent lipid peroxidation system, the potential effects of trisulfide appear to be mediated via enzyme inhibition. These results suggest that pretreatment of the trisulfide derivatives may affect the toxic function of exogenous xenobiotics or drugs, which are reduced by the liver enzyme cytochrome P-450 to radical species.

ESR and HPLC-EC analysis of ethanol oxidation to 1-hydroxyethyl radical: rapid reduction and quantification of POBN and PBN nitroxides

Extensive ESR spin-trapping studies have shown that ethanol is oxidized to 1-hydroxyethyl radical (HER) by rat and deer mice liver microsomal systems. The ESR detection of POBN/HER nitroxide in bile, and formation of antibodies, which recognize HER adducts in alcoholics, suggest that HER is produced in vivo. In liver, where ethanol is primarily metabolized, only traces of PBN/HER nitroxide are documented. One limitation of the ESR spin-trapping technique, however, is that the nitroxides formed in the presence of cellular reductants can be metabolized to the corresponding ESR “silent” hydroxylamines. Ascorbate and NADPH plus liver microsomes were found to reduce rapidly both POBN/HER and PBN/HER nitroxides to their ESR “silent” hydroxylamine derivatives. An HPLC method with electrochemical detection was developed for the detection and quantification of both POBN/HER and PBN/HER nitroxides, as well as their hydroxylamines. Both the diastereomers of the POBN/HER nitroxide and hydroxylamine can be detected, as can both isomers of the PBN/HER nitroxide, and it is estimated that the sensitivity of the HPLC procedure is in the nM range when using EC detection. The hydroxylamines are stable in ethanol, while pH-dependent auto-oxidation occurs in aqueous buffers. Some of the characteristics associated with HER formation by microsomes as detected with ESR (e.g., sensitivity to SOD and catalase, increase after induction of CYP2E1) are reproduced with the HPLC method. By quantification of the POBN/HER hydroxylamine, the NADPH-dependent rates of HER formation by microsomes from pyrazole-treated rats are estimated to be about 1–1.5 nmol HER per min per mg microsomal protein. This rate is less, as compared to the two electron-dependent rate of acetaldehyde formation by these microsomes, about 10–15 nmol per min per mg protein. Thus, at first approximation, the one electron-dependent rate of ethanol oxidation is about 10% the two electron-dependent rate by isolated microsomes. The HPLC procedure can readily detect the POBN/HER and PBN/HER nitroxides and their hydroxylamine derivatives in the same sample and may be of value in detecting HER spin-trapped adducts under biological reducing conditions.

Diaminotoluenes induce intrachromosomal recombination and free radicals in Saccharomyces cerevisiae

The carcinogenicity of aniline-based aromatic amines is poorly reflected by their activity in short-term mutagenicity assays such as the Salmonella typhimurium reverse mutation (Ames) assay. More information about the mechanism of action of such carcinogens is needed. Here we report the effects on DEL recombination in Saccharomyces cerevisiae of the carcinogen 2,4-diaminotoluene and its structural isomer 2,6-diaminotoluene, which is reported to be non-carcinogenic. Both compounds are detected as equally mutagenic in the Salmonella assay. In the absence of any external metabolizing system both compounds were recombinagenic in the DEL assay, with the carcinogen being a more potent inducer of deletions than the non-carcinogen. In the presence of Aroclor-induced rat liver S9, however, the carcinogen 2,4-diaminotoluene became a 2-fold more potent inducer of deletions, and the non-carcinogen 2,6-diaminotoluene was rendered less toxic and no induced recombination was observed. 2,4-Diaminotoluene is distinguished from its non-carcinogen analog in the DEL assay, therefore, on the basis of a preferential activation of the carcinogen in the presence of a rat liver microsomal metabolizing system. Free radical species are produced by several carcinogens and have been implicated in carcinogenesis. We further investigated whether exposure of yeast to either 2,4-diaminotoluene or 2,6-diaminotoluene resulted in a rise in intracellular free radical species. The effects of the free radical scavenger N-acetylcysteine on toxicity and recombination induced by the two compounds and intracellular oxidation of the free radical-sensitive reporter compound dichlorofluorescin diacetate were studied. Both 2,4- and 2,6-diaminotoluene produced free radical species in yeast, indicating that the reason for the differential activity of the compounds for induced deletions is not reflected in any difference in the production of free radical species.

Inhibition of lipid peroxidation and the active oxygen radical scavenging effect of anthocyanin pigments isolated from Phaseolus vulgaris L

No attention has been paid to anthocyanin pigments from the viewpoint of inhibitors of lipid peroxidation and scavengers of active oxygen radicals; therefore, we investigated the antioxidative, radical scavenging, and inhibitory effects on lipid peroxidation by UV light irradiation of three anthocyanin pigments, pelargonidin 3-O-β- d-glucoside (P3G), cyanidin 3-O-β- d-glucoside (C3G), and delphinidin 3-O-β- d-glucoside (D3G), isolated from the Phaseolus vulgaris L. seed coat, and their aglycons, pelargonidin chloride (Pel), cyanidin chloride (Cy), and delphinidin chloride (Del). All pigments had strong antioxidative activity in a liposomal system and reduced the formation of malondialdehyde by UVB irradiation. On the other hand, the extent of antioxidative activity in a rat liver microsomal system and the scavenging effect of hydroxyl radicals (·OH) and Superoxide anion radicals (O 2 −) were influenced by their own structures.

GlutathioneS-Transferase-Catalyzed Conjugation of Bioactivated Aflatoxin B1in Rabbit Lung and Liver

Aflatoxin B1(AFB1) requires bioactivation to AFB1-8,9-epoxide for carcinogenicity, and glutathioneS-transferase (GST)-catalyzed conjugation of activated AFB1with glutathione (GSH) is a critical determinant of susceptibility to the mycotoxin. Incubations containing [3H]AFB1, rabbit liver microsomes, an NADPH-generating system, 1 mMGSH, and GST-containing lung or liver cytosol were performed to assess the abilities of lung and liver GSTs to conjugate AFB1-8,9-epoxide. [3H]AFB1–GSH was isolated by isocratic reverse-phase high-performance liquid chromatography (HPLC) and quantitated by liquid scintillation spectroscopy. Maximal [3H]AFB1–GSH formation rates were significantly lower for lung than for liver (0.3 ± 0.1 and 1.7 ± 0.4 nmol/mg/hr, respectively). Immunoprecipitation of rabbit pulmonary cytosolic GSTs with anti-alpha or anti-mu GST antisera decreased [3H]AFB1–GSH production by approximately 45 and 51%, respectively, indicating that alpha-class and mu-class GSTs are of similar importance in catalyzing this reaction in the lung. Because mu-class GSTs comprise only a small proportion of total lung GST content, these enzymes have high specific activity toward AFB1-8,9-epoxide. In contrast, the pi-class GST appeared to play a negligible role. Using a rat liver microsomal system to generate both AFB1exo- andendo-epoxide isomers, and analysis based on chiral HPLC, we found that rabbit liver cytosolic GSTs catalyzed formation of both AFB1exo- andendo-epoxide–GSH conjugates, whereas pulmonary cytosolic GSTs catalyzed formation of only theexostereoisomer at detectable levels. Despite a preference for conjugating the more mutagenic AFB1exo-epoxide isomer, the relatively low capacity for GST-catalyzed detoxification of bioactivated AFB1in lung may be an important factor in the susceptibility of the lung to AFB1toxicity.

Application of chemical P-450 model systems to studies on drug metabolism. Part X. Novel hydroxylactonization of γ,δ- and β,γ- unsaturated carboxylic acids with an iron porphyrin–iodosylbenzene system

The oxidative hydroxylactonization of γ,δ- and β,γ-unsaturated carboxylic acids by a chemical cytochrome P-450 model and rat liver microsomal systems has been investigated. In the chemical system using meso-tetrakis(2,6-dichlorphenyl)porphyrin iron chloride [Fe(TDCIPP)Cl] with iodosylbenzene (PhIO), γ,δ-unsaturated carboxylic acids have been converted into δ-hydroxy-γ-lactones in high yield and with high stereoselectivity. As an example of a β,γ-unsaturated carboxylic acid, indomethacin has been converted into the corresponding β-hydroxy γ-lactone. Several experiments directed toward mechanistic elucidation of the lactonization exclude a mechanism occurring via an epoxide intermediate. The products have been used as standards to identify the metabolites in the microsomal oxidation. In the case of indomethacin, the γ-lactone form is detected as a metabolite in the rat liver microsomal system, in a yield of 1.33%; the yield is significantly decreased in the presence of 2-diethylaminoethyl-2,2-diphenylvalerate hydrochloride (SKF-525A) and under a mixed CO–O2(4:1) atmosphere. Thus, these metabolites are considered to be formed by a cytochrome P-450-dependent reaction.

Effects of erythromycin and roxithromycin on oxidation of testosterone and nifedipine catalyzed by CYP3A4 in human liver microsomes.

Roxithromycin and erythromycin were incubated with rat and human liver microsomal or reconstituted cytochrome P450 (P450 or CYP) monooxygenase systems in the presence of an NADPH-generating system, and the effects of these chemicals on testosterone 6 beta-hydroxylation and nifedipine oxidation activities were compared with those of typical CYP3A4 inhibitors including ketoconazole, troleandomycin, and gestodene. Roxithromycin and erythromycin were found to be relatively weak inhibitors of testosterone 6 beta-hydroxylation and nifedipine oxidation activities by rat and human liver microsomes or by reconstituted systems containing CYP3A4/5. Formation of an inhibitory P450-metabolite complex was determined spectrally by incubating troleandomycin with human liver microsomes; the extents of the complex formation were lesser in liver microsomes of humans than those of rats treated with dexamethasone. Erythromycin and roxithromycin were also activated slightly by rat liver microsomes to form P450.Fe(II)-metabolite complex, although these chemicals caused very little or undetectable levels, respectively, of spectral changes by human liver microsomes even when a human sample which contained relatively high levels of CYP3A4 was used. These results suggested that roxithromycin and erythromycin were relatively less potent to inhibit CYP3A4-catalytic activities in human liver microsomes, because of their low capabilities to form P450.Fe(II)-metabolite complex.

L-519, a phenolic compound, inhibits metabolism of benzo(a)pyrene and mutagenesis induced by benzo(a)pyrene

L-519 is a phenolic compound. In this study, L-519 was found to inhibit the mutagenicity of benzo(a)pyrene [B(a)P] in Salmonella Typhimurium TA 97 and TA 100 and inhibit the cytochrome P-450 mediated metabolism of B(a)P. It also inhibited the epidermal ODC activity induced by croton oil. L-519 also decreased the lipid peroxidation induced by FeSO4 and Cysteine in rat liver microsome system. Our results demonstrated that L-519 exhibited anti-mutagenicity, anti-initiation as well as anti-promotion activities.

Application of chemical cytochrome P-450 model systems to studies on drug metabolism—VIII. Novel metabolism of carboxylic acids via oxidative decarboxylation

The oxidative decarboxylation of carboxylic acids by the chemical cytochrome P-450 model and rat liver microsomal systems was investigated. In the chemical system using meso-tetrakis(pentafluorophenyl)porphyrin iron chloride [Fe(TPFPP)Cl] with iodosylbenzene (PhIO), α-arylcarboxylic acids and α,α,α-trisubstituted acetic acids are converted to the corresponding one-carbon-reduced alcohol ( I) and carbonyl derivatives ( II) via oxidative decarboxylation. These products were then used as standards to identify the metabolites in vivo and in vitro. Biliary excretion of I a and II a in bile duct-cannulated rats after oral administration of ketoprofen amounted to 0.22 and 0.03% of the dose, respectively. In the case of indomethacin, I b and II b were detected as metabolites in the rat liver microsomal system, in yields of 2.8 and 0.29%, respectively. Further, the yields of I b and II b were decreased in the presence of SKF-525A. Thus, these metabolites were formed by cytochrome P-450-dependent reactions. Metabolites I a , I b, II a and II b had moderate to strong inhibitory activities on arachidonic acid-induced platelet aggregation and cyclooxygenase activity in vitro, comparable to those of the parent compounds.

Antioxidative Activity of the Anthocyanin Pigments Cyanidin 3-O-.beta.-D-Glucoside and Cyanidin

The antioxidative activity of the anthocyanin pigments cyanidin 3-O-β-D-glucoside (C3G) and cyanidin (Cy) was examined by using linoleic acid autoxidation, liposome, rabbit erythrocyte membrane, and rat liver microsomal systems. C3G and Cy had antioxidative activity in all systems. Cy had a stronger activity than C3G and the same activity as α-tocopherol (Toc) in the liposome and rabbit erythrocyte membrane systems. In the rat liver microsomal system, Cy and C3G exhibited stronger activity than Toc. These data suggest that the pigments may play an important role in the prevention of lipid peroxidation of cell membranes induced by active oxygen radicals in living systems as dietary antioxidants after ingestion

Mutagenic evaluation of primaquine, pentaquine and pamaquine in the Salmonella/mammalian microsome assay

The 8-aminoquinolines, primaquine, pentaquine and pamaquine, were investigated for mutagenic activity in Salmonella typhimurium strains TA100, TA98, TA97 and TA102 in the rat liver microsomal activation system. Primaquine and pentaquine induced mutations in TA97 in the presence and absence of S9 mix. Pamaquine was mutagenic to TA98 only in the absence of S9 mix.

Copper-Metallothionein from the Toxic Milk Mutant Mouse Enhances Lipid Peroxidation Initiated by an Organic Hydroperoxide

The toxic milk mutation is an autosomal recessive mutation found in an inbred C57BL/6J strain of mice which results in an excessive hepatic accumulation of copper (Cu), mostly associated with metallothionein (MT). The possible toxicological significance of elevated levels of hepatic copper-metallothionein (Cu-MT) was assessed. The effects of Cu-MT on lipid peroxidation (LP) initiated by an organic peroxide were investigated in an in vitro rat liver microsomal incubation system. Addition of Cu-MT (3 μM) could significantly enhance (100%) LP induced by addition of tertiary-butyl hydroperoxide (t-BHP, 0.1 mM). Similar incubations of Cu-MT in the absence of t-BHP showed negligible LP. Addition of copper sulfate at high concentrations (100 μM) also increased t-BHP induced LP, but the enhancement was less pronounced than observed with Cu-MT addition. Chelation of copper with bovine serum albumin and triethylenetetramine tetrahydrochloride eliminated the enhancement of LP by Cu-MT. Evidence is provided on degradation of MT and release of free Cu in the incubation system. Additions of deferoxamine were also found to prevent LP. Therefore, chelatable Cu, released from Cu-MT, may be responsible for the enhancement of the iron-dependent LP in this system but Cu-MT alone in the absence of iron cannot initiate LP. These in vitro results suggest that conditions resulting in high cellular levels of Cu-MT may exhibit a predisposition to oxidative stress.

Suppressive Effect of Astaxanthin on Lipid Peroxidation Induced in Rats.

Astaxanthin, one of the naturally occurring derivatives of vitamin A, was found to suppress lipid peroxidation in a rat liver microsomal system. This substance was also found to suppress lipid peroxidation in rats induced by either carbon tetrachloride or 60Co-irradiation. The suppressive effect of astaxanthin on lipid peroxidation was more potent than that of α-tocopherol.