Species differences in activating and inactivating enzymes related to the control of mutagenic metabolites

Microsomal monooxygenases catalyze the biosynthesis of epoxides from olefinic and aromatic compounds whilst microsomal epoxide hydratase and cytoplasmic glutathione S-transferases are responsible for their further biotransformation. Although catalytically very efficient the cytoplasmic glutathione S-transferases play, due to their subcellular localization, a minor role in the inactivation of epoxides derived from large lipophilic compounds and were, therefore, not included in this study. It was shown with such a lipophilic compound, benzo(a)pyrene, as a model substance and with liver enzyme mediated bacterial mutagenesis as biological endpoint that species and strain differences in epoxide hydratase and monooxygenases are reflected in very dramatic differences in mutagenicity of benzo(a)pyrene which varied from extremely potent to a degree which could easily be overlooked. In order to investigate whether the differences in enzyme activities were causally linked to the observed differences in mutagenicity, the enzyme activities were modulated by inhibition and induction. These manipulations were always accompanied by the corresponding changes in mutagenicity.It is concluded that species such as mice which possess high monooxygenase activity but very low epoxide hydratase activity are much more susceptible than man to those toxic effects which are mediated by metabolically formed epoxides which are substrates of epoxide hydratase. In this regard, it is especially noteworthy that mice possess a much lower hepatic epoxide hydratase activity than man.Presented at the Symposium Influence of Metabolic Activations and Inactivations on Toxic Effects held at the 18th Spring Meeting of the Deutsche Pharmakologische Gesellschaft, Section Toxicology, D-6500 Mainz, March 15, 1977     

Dual role of epoxide hydratase in both activation and inactivation of benzo(a)pyrene

The effect of epoxide hydratase upon the mutagenicity of benzo(a)pyrene was investigated using two Salmonella typhimurium strains (TA 1537 and TA 98). These two bacterial strains were found to differ characteristically in their susceptibility to different mutagens biologically produced from benzo(a)pyrene providing a diagnostic tool to investigate which types of mutagenic metabolites were produced in various metabolic situations. The results showed that the pattern of mutagenic metabolites produced by microsomes from methylcholanthrene-treated mice was very different from that produced by microsomes from phenobarbital-treated or untreated mice. However in all cases at least two mutagenic metabolites were produced. Epoxide hydratase was very efficient at reducing the mutagenic effect when benzo(a)pyrene was activated by microsomes from untreated or phenobarbital-treated mice. However, when microsomes from methylcholanthrene-treated mice were used the effect of hydratase depended upon the benzo(a)pyrene concentration. At low concentrations the mutagenicity was increased by addition of epoxide hydratase and decreased by inhibition of the hydratase. At high concentrations the reverse was true. These findings indicate that when microsomes from untreated and phenobarbital-treated mice were used the main contributors to the mutagenicity were simple epoxides (or compounds arising non-enzymically from them). The activation of dihydrodiols must, however, contribute to a significant extent when microsomes from methylcholanthrene-treated mice were used. Thus the role of epoxide hydratase was determined by the monooxygenase form present in the microsomes in the activating system.Presented at the Symposium Influence of Metabolic Activations and Inactivations on Toxic Effects held at the 18th Spring Meeting of the Deutsche Pharmakologische Gesellschaft, Section Toxicology, D-6500 Mainz, March 15, 1977     

Covalent glutathione conjugation to cyanobacterial hepatotoxin microcystin LR by F344 rat cytosolic and microsomal glutathione S-transferases

Most investigators report that microcystins (MCs) bound covalently to SH compounds, such as L-cysteine and reduced glutathione (GSH). However, their studies were based on a high pH condition. In the present study, we investigated the reaction between microcystin LR (MCLR) and GSH in various pH conditions. As a result, we found that no MCLR conjugated with GSH in these conditions, and MCLR mixed with GSH showed different peaks of retention time compared with intact MCLR on the high performance liquid chromatography-mass spectrometry (LC-MS) chromatograms. Furthermore, we found the GSH conjugate of MCLR was detected in the glutathione S-transferase (GST) assay using F344 rat liver cytosol and microsomes. This indicates that the covalent GSH conjugation was caused only by an enzymatic activity. We conclude, therefore, that the reaction is caused by enzymatic action and is not due to the Michael reaction.     

Exposure to epichlorohydrin and dimethylformamide, glutathione S-transferases and sister chromatid exchange frequencies in peripheral lymphocytes

Workers in epoxy resin, synthetic leather, and printed circuit board manufacturing plants are exposed to epichlorohydrin (ECH), or dimethylformamide (DMF), or both. ECH, an alkylating agent, has been shown to cause malignancy in animals, but its genotoxicity in humans is unclear. DMF is a well-known hepatotoxic chemical, although evidence of its genotoxicity in humans is also limited. In this study, we examined the effects of exposure to ECH and DMF on sister chromatid exchange (SCE) in plant workers, in order to examine the genotoxicity of these two agents. Because the genotoxicity of certain agents can be modulated by metabolic traits, we also investigated influence of the glutathione S-transferase (GST) μ (GST M1) and GST θ (GST T1) genes on the genotoxicity of ECH and DMF. A total of 85 male plant workers were included in this study. The subjects were divided into five exposure groups, based on their job titles and the airborne ECH and DMF concentrations in their areas of work. A questionnaire was administered to obtain detailed occupational, smoking, alcohol consumption, and medication histories. Standardized cytogenetic methods were used to determine the frequency of sister chromatid exchange (SCE) in peripheral blood lymphocytes. GST M1 and GST T1 genotypes were identified using polymerase chain reaction (PCR). In analysis, smoking was significantly associated with increased SCE frequency (P < 0.01). Workers with high ECH exposure also had significantly higher SCE frequencies than those with low or no ECH exposure (P < 0.05). However, DMF exposure was not associated with SCE frequency. The GST M1 null genotype was also found to be associated with an increased SCE frequency (P = 0.06). We conclude that ECH exposure may be associated with genetic toxicity and that DMF does not appear to be genotoxic. Received: 24 November 1998 / Accepted: 9 March 1999     

Screening of Lactobacillus strains for their ability to bind Benzo(a)pyrene and the mechanism of the process

In order to investigate the binding ability of Lactobacillus strains to Benzo(a)pyrene (BaP), 15 strains were analysed. L. plantarum CICC 22135 and L. pentosus CICC 23163 exhibited high efficiency in removing BaP from aqueous medium; the binding rates were 66.76% and 64.31%, respectively. This process was affected by temperature, incubation time and pH, and cell viability was not necessary for the binding ability. Additionally, both strains, especially strain CICC 23163 showed high specificity in binding BaP. The cell-BaP complexes were stable in aqueous medium. The mechanism of binding was investigated by examining the binding ability of different components of the microorganism cells. The results revealed that peptidoglycans played an important role in binding BaP and its structural integrity was required. Consequently, we proposed that the mechanism of this process was a physisorption and peptidoglycan was the main binding site. These two strains may be used for dietary detoxification in human diet and animal feed.     

Enzyme-mediated dichloromethane toxicity and mutagenicity of bacterial and mammalian dichloromethane-active glutathione S-transferases

The kinetic properties of bacterial and rat liver glutathione S-transferases (GST) active with dichloromethane (DCM) were compared. The theta class glutathione S-transferase (rGSTT1-1) from rat liver had an affinity for dihalomethanes lower by three orders of magnitude (K _app > 50 mM) than the bacterial DCM dehalogenase/GST from Methylophilus sp. DM11. Unlike the bacterial DCM dehalogenase, the rat enzyme was unable to support growth of the dehalogenase minus Methylobacterium sp. DM4-2cr mutant with DCM. Moreover, the presence of DCM inhibited growth with methanol of the DM4-2cr transconjugant expressing the rat liver GSTT1-1. In Salmonella typhimurium TA1535, expression of rat and bacterial DCM-active GST from a plasmid in the presence of DCM yielded up to 5.3 times more reversions to histidine prototrophy in the transconjugant expressing the rat enzyme. Under the same conditions, however, GST-mediated conversion of DCM to formaldehyde was lower in cell-free extracts of the transconjugant expressing the rat GSTT1 than in the corresponding strain expressing the bacterial DCM dehalogenase. This provided new evidence that formaldehyde was not the main toxicant associated with GST-mediated DCM conversion, and indicated that an intermediate in the transformation of DCM by GST, presumably S-chloromethylglutathione, was responsible for the observed effects. The marked differences in substrate affinity of rat and bacterial DCM-active GST, as well as in the toxicity and genotoxicity associated with expression of these enzymes in bacteria, suggest that bacterial DCM dehalogenases/GST have evolved to minimise the toxic effects associated with glutathione-mediated catalysis of DCM conversion. Received: 14 September 1998 / Accepted: 11 January 1999     

Pentachlorophenol enhances 9-hydroxybenzo [a] pyrene-induced hepatic DNA adduct formation in vivo and inhibits microsomal epoxide hydrolase and glutathione S-transferase activities in vitro: likely inhibition of epoxide detoxication by pentachlorophenol

 We recently reported that co-administration to female mice of tamoxifen or 4-hydroxytamoxifen (4-OH-tamoxifen) with pentachlorophenol (PCP), but not with 2,6-dichloro-4-nitrophenol (DNCP) results in strong intensification of a specific subgroup, termed group I, of tamoxifen-DNA adducts in female mouse liver. As both PCP and DCNP are sulfotransferase inhibitors, we concluded that the intensification of tamoxifen group I adducts is probably not due to inhibition of sulfation by these phenols of a tamoxifen metabolite. Since epoxide derivatives of 4-OH-tamoxifen are potential candidates involved in tamoxifen-induced DNA damage, the hypothesis was developed and tested that PCP inhibits epoxide detoxication. As 4-OH-tamoxifen metabolites were unavailable to us, we employed indirect approaches to test this hypothesis. In the first set of experiments we determined whether PCP would augment DNA adduct formation from the benzo[a]pyrene metabolite, 9-hydroxybenzo[a]pyrene (9-OH-BP), as 9-OH-BP-4,5-epoxide is known to be involved in the metabolic activation of this compound. Female mice were given a single i.p. dose of 9-OH-BP (50 μmol/kg) either alone or in combination with PCP (75 μmol/kg), and hepatic DNA adducts were measured 24 h later by nuclease P1-enhanced bisphosphate ³²P-postlabeling. Co-administration of PCP with 9-OH-BP resulted in a statistically significant 1.5- to 1.7-fold increase in 9-OH-BP adduct levels versus 9-OH-BP controls. In order to determine whether PCP inhibits the enzymatic detoxication of epoxides in vitro, in a second set of experiments, the effects of PCP on liver microsomal epoxide hydrolase (mEH) and purified equine liver glutathione S-transferase (GST) activities were studied using, respectively, styrene-7,8-oxide and 1-chloro-2,4-dinitrobenzene (CDNB) as substrates. Incubation of mouse liver microsomes with PCP (10–100 μM) strongly inhibited (by 21–97%) mEH activity in a dose-dependent manner, the IC₅₀ being 35 μM. DCNP was ineffective as a mEH inactivator. PCP also inhibited purified equine liver GST activity, with an IC₅₀ of 23.5 μM. Taken together, the results of this study strongly support the hypothesis that PCP inhibited enzymatic detoxication of epoxides in vivo and in vitro. By this mechanism PCP would lead to enhancement of DNA damage caused by 9-OH-BP, and possibly other drugs and their metabolites, which undergo epoxidation prior to DNA binding. Received: 28 November 1995/Accepted: 12 March 1996     

Utilizing proteomic approach to identify nuclear translocation related serine kinase phosphorylation site of GNMT as downstream effector for benzo[a]pyrene

Glycine N-methyltransferase (GNMT) protein is highly expressed in certain tissues, such as liver, pancreas, and prostate. GNMT serves multiple roles which include a methyl group transfer enzyme and a liver tumor suppressor. Benzo(a)pyrene (BaP), a family member of polycyclic aromatic hydrocarbon (PAH), is a known environmental carcinogen found in coal tar, tobacco smoke, barbecued food and incomplete combustion of auto fuel. BaP recruits cytochrome P450 to transform itself into benzo(a)pyrene-7,8-diol-9,10-epoxide (B(a)PDE), which covalently interacts with DNA causing tumorigenesis. BaP can be detoxified through GNMT and induces GNMT translocation into the cellular nucleus. GNMT translocation is accompanied by phosphorylation, but the role of phosphorylation in GNMT remains to be explored. Using liquid chromatography coupled with tandem mass spectrometry, this study identified serine 9 of GNMT as the phosphorylation site upon BaP treatment. When serine 9 was mutated and lost the capability to be phosphorylated, the occurrence of BaP-induced GNMT nuclear translocation was dramatically decreased. Also, this mutant from of GNMT lost the ability of phosphorylation and increased cytochrome P450 1A1 (Cyp1a) expression upon BaP treatment. In addition, protein kinase C (PKC) and c-Jun NH2-terminal kinase (JNK) may be required for such phosphorylation. Further characterization of phosphorylated GNMT for its link to BaP may bring new insights into chemical detoxification.     

Measurement of hemoglobin and albumin adducts of benzo(a)pyrenediolepoxide and their rate of elimination in the female Sprague-Dawley rat

A high performance liquid chromatographic technique coupled with fluorometric detection was used to study the disappearance rate of the hemoglobin and albumin adducts of benzo(a)pyrene (BaP) in female Sprague-Dawley rats. The technique was considered adequate based on good recovery (>80%) of the analyte and satisfactory intra-assay coefficients of variations of 7.8% and 4.2% for hemoglobin and albumin adducts, respectively. The persistence of the adducts was determined in rats injected intravenously with 550 nmol BaP diolepoxide, the electrophilic metabolite of BaP. In a first experiment the isolated protein fractions were directly subjected to mild acid hydrolysis to yield the ultimate analyte, BaP tetrol. In a second experiment these fractions were first digested by proteases in order to liberate any unbound lipophilic BaP tetrol possibly hidden in the hydrophobic portions of the protein matrices. It was found that 70–100% of the measured tetrols originated from true adducts. The half-life of the free tetrol was 2.8 and 1.6 days in the hemoglobin and albumin fractions, respectively, compared to 10.7 and 3.6 days for the true adducts. Based on the total amount of tetrols, the mean half-life of the latter in the hemoglobin and albumin fractions determined from the two experiments was 9.0 and 2.7 days, respectively, assuming first order kinetics. The greater persistence of the hemoglobin adduct compared to that of the albumin adduct is coherent with the biological half-life of the intact proteins. However, the observed disappearance profile obtained does not fit with current models of adduct formation and removal.     

Comparison of benzo(a)pyrene metabolism by testicular homogenate and the isolated perfused testis of rat following 2,3,7,8-tetrachlorodibenzo-P-dioxin treatment

Benzo(a)pyrene (BP) metabolism was studied in the cell free testicular homogenate and in the isolated perfused rat testis 72 h following tetrachlorodibenzo-P-dioxin (TCDD). The BP concentration for both metabolic systems was 2 × 10^–7 M. BP metabolites were extracted from testicular homogenate, perfusate and testicular tissue and subjected to high-pressure liquid Chromatographic analysis. The ratio of various BP metabolites in the cell free homogenates ranged from 3.5 to 164 times those of the isolated perfused testis, and the total BP metabolites in the cell free system of either control or TCDD-induced testis were 16 times that of the intact isolated perfused testis. The major BP metabolites in the organic extractable phase from the isolated perfused testis and the testicular homogenate were BP dihydrodiols and BP phenols, respectively.The ratio of water soluble metabolites to organic soluble metabolites in the homogenate and the isolated perfused testis is 1.1 and 3.0 respectively. Therefore, in the intact isolated testis, water soluble BP metabolites are formed three times greater than those of the organic soluble BP metabolites, and thus suggests that specific conjugating enzyme activities in the intact testis are greater than those of the homogenates. The magnitude of various BP metabolites in either the homogenates or the isolated perfused testis of TCDD treated rats ranged from 1.4 to 2.2 times their respective controls, except 4,5-dihydroxy-4,5-dihydrobenzo(a)pyrene in the isolated perfused testis was not altered by TCDD treatment. In conclusion, the isolated perfused rat testis is metabolically active and capable of biotransforming PAH. This system better reflects metabolic capability of the intact organ in the rats than do cell free homogenate system. The isolated perfused testis system retains the integrity of the complex biological organization of tissues, cell types, and enzymes in testicular metabolism and may provide data that may aid in the prediction of germ cell mutation as well as toxicity.The Abbreviations Used are PAH polycyclic aromatic hydrocarbons - BP benzo(a)pyrene - 9,10-diol 9,10-dihydroxy-9,10-dihydrobenzo(a)pyrene - 7,8-diol 7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene - 4,5-diol 4,5-dihydroxy-4,5-dihydrobenzo(a)pyrene - 3-OH 3-hydroxybenzo(a)pyrene - 9-OH 9-hy-droxybenzo(a)pyrene - 7-OH 7-hydroxybenzo(a)pyrene - 12-OH 12-hydroxybenzo(a)pyrene - 1,6-quinone benzo(a)pyrene-1,6-dione - 3,6-quinone benzo(a)pyrene 3,6-dione - 6,12-quinone benzo(a)pyrene 6,12-dione - DMBA 7,12-dimethylbenzanthracene - DMN dimethylnitrosamine - 7,8-diol 9,10-epoxide 5,7-t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo(a)pyrene - HPLC high pressure liquid chromatography - TCDD 2,3,7,8-tetrachlorodibenzo-P-dioxin - HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid - NADP⁺ nicotinamide adenine dinucleotide - AHH aryl hydrocarbon hydroxylase - EH epoxide hydrolase - GSH-T glutathione transferase - HPRT hypoxanthine phosphoribosyltransferase     

Inhibition of human glutathione S-transferases by curcumin and analogues

1. Glutathione S-transferases (GSTs) are important phase II drug-metabolizing enzymes that play a major role in protecting cells from the toxic insults of electrophilic compounds. Curcumin, a promising chemotherapeutic agent, inhibits human GSTA1-1, GSTM1-1, and GSTP1-1 isoenzymes.

2. In the present study, the effect of three series of curcumin analogues, 2,6-dibenzylidenecyclohexanone (A series), 2,5-dibenzylidenecyclopentanone (B series), and 1,4-pentadiene-3-one (C series) substituted analogues (n?=?34), on these three human GST isoenzymes, and on human and rat liver cytosolic GSTs, was investigated using 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate.

3. Most of the 34 curcumin analogues showed less potent inhibitory activities towards GSTA1-1, GSTM1-1, and GSTP1-1 than the parent curcumin. Compounds B14 and C10 were the most potent inhibitors of GSTA1-1 and human liver cytosolic GSTs, with IC₅₀ values of 0.2–0.6 μM. The most potent inhibitors of GSTM1-1 were C1, C3 and C10, with IC₅₀ values of 0.2–0.7 μM. Similarly, GSTP1-1 was predominantly strongly inhibited by compounds of the C series C0, C1, C2 C10 and A0, with IC₅₀ values of 0.4–4.6 μM. Compounds in the B series showed no significant inhibition of GSTP1-1.

4. Molecular Operating Environment (MOE) program-based quantitative structure–activity relationship (QSAR) analyses have also suggested the relevance of Van der Waals surface area and compound lipophilicity factors for the inhibition of GSTA1-1 and GSTM1-1 and partial charge factors for GSTP1-1. These results may be useful in the design and synthesis of curcumin analogues with either more or less potency for GST inhibition.

     

DNA damage caused by benzo(a)pyrene in MCF-7 cells is increased by verapamil, probenecid and PSC833

The aim of this study was to clarify whether pharmaceutical drugs capable of inhibiting ABC-transporters affect the toxicity of benzo(a)pyrene (BP). MCF-7 breast adenocarcinoma cells were cultured for 24 and 48 h with benzo(a)pyrene (1 microM) and the transporter inhibitors verapamil (0.125-100 microM), PSC833 (0.05-5 microM) or probenecid (0.05-2 mM). DNA binding of benzo(a)pyrene-7,8-diol-9,10-epoxide (BPDE) was analyzed by synchronous fluorescence spectrophotometry and p53 protein by immunoblotting. BP metabolism was studied using thin layer chromatography (TLC). MTT assay and ATP quantitation were used for the analysis of cell viability. At 24 h there was no statistically significant increase in the DNA-adduct formation by any of the used inhibitors. However, at 48 h all of the inhibitors, in concentrations known to effectively block ABC transporters, increased the BPDE-DNA adduct formation 1.5 to 2-fold compared to adduct formation with BP only. PSC833 and verapamil also increased p53 protein expression at 48 h (p<0.05). Probenecid decreased glucuronidation of (3)H-BP metabolites. Other inhibitors did not decrease statistically significantly the overall formation of water-soluble metabolites. BP alone slightly decreased viability of cells at 48 h according to ATP quantitation as compared to vehicle treated controls (86.4+/-16.4%). Even though the used inhibitors showed some cytotoxicity, the combination of BP and inhibitors did not decrease cell viability in synergistic manner. According to these results certain pharmaceutical drugs may increase DNA damage caused by benzo(a)pyrene in MCF-7 cells at least partly through the inhibition of transporters. Taking into account the complex metabolism of BP and lack of specificity of the inhibitors used, it is likely that increased DNA damage seen in this study was the result of multiple interactions between the inhibitors, BP metabolism and the efflux of the compounds.     

Depletion of mitochondrial enzyme system in liver,lung, brain,stomach and kidney induced by benzo(a)pyrene

Mitochondrial dysfunction has recently received considerable attention as it plays an important role in adult human pathology caused by various drugs, endogenous agents and environmental agents. Benzo(a)pyrene (BaP), is a ubiquitous environmental contaminant mainly derived from anthropogenic activity during incomplete combustion of organic materials from various sources. The present study aimed to evaluate the effects of benzo(a)pyrene (BaP) on mitochondrial enzymes in the multiple organs including liver, lung, brain, stomach and kidney. ICR mice were exposed to different doses of BaP (2.5, 5 and 10 mg/kg body weight) through oral gavage and intraperitoneal injection treatment for 13 weeks consecutively. The induced mitochondrial damage in the examined organs was assayed in terms of significant increase in lipid peroxidation (LPO) and prominent decrease in antioxidant enzymes. Non enzymatic antioxidants and Krebs cycle’s enzymes were also significantly decreased in mitochondria. Additionally, BaP induced the body growth retardation and decrease in relative liver weight, increase in relative lung, stomach, kidney and brain weights, and this was further certified through histopathological lesions. Liver and lungs were more prominently damaged by BaP. The mitochondrial depletion increased in BaP dose-dependent manner.     

Influence of estradiol on the benzo(a)pyrene hydroxylase activity induced by hexachlorocyclohexane

Basal BP-hydroxylase activity was measured in male Swiss mice from the age of 3 weeks to 20 months. Maximal enzyme activity was at the age of 5 months. Comparison of the inducibility of BP-hydroxylase by HCH was also investigated in male and female mice of different ages. Male mice showed higher induction of BP-hydroxylase by HCH than females of the same ages. Sterilization of female mice enhanced enzyme induction. Estradiol exhibited competitive inhibition of BP-hydroxylase activity. After treatment with HCH for 8 months, female mice had a lower tumour incidence than males, and this paralleled a lower induction of BP-hydroxylase.     

Combined effects of curcumin and piperine in ameliorating benzo(a)pyrene induced DNA damage

The present study was planned to investigate the antigenotoxic effects of curcumin and piperine separately and in combination against benzo(a)pyrene (BaP) induced DNA damage in lungs and livers of mice. Male Swiss albino mice received curcumin (100 mg kg⁻¹ body weight) and piperine (20 mg kg⁻¹ body weight) separately as well as in combination orally in corn oil for 7 days as pretreatments and subsequently, 2 h after, BaP was administered orally in corn oil (125 mg kg⁻¹ body weight). A single dose of BaP to normal mice increased the level of 8-oxo-2′-deoxyguanosine (8-oxo-dG) content and % DNA in the comet tail in the lungs and liver. Pretreatments of curcumin and curcumin plus piperine before administration of single dose of BaP significantly decreased the levels of 8-oxo-dG content and % DNA in the comet tail in both the tissues. Moreover, the genoprotective potential of curcumin plus piperine was significantly higher as compared to curcumin alone against BaP induced DNA damage.     

Transfer of benzo(a)pyrene into mouse embryos and fetuses

The distribution of radioactive material within maternal and embryonic/fetal mouse tissue was studied over a period of 2 days following a single dose of¹⁴C-benzo (a)pyrene (B(a)P) orally on days 11, 12, 13 or 18 of pregnancy. B(a)P poorly penetrated into the embryo/fetus, and¹⁴C-radioactivity was found in embryonic tissues at concentrations one to two orders of magnitude lower than in maternal organs. If the compound was administered for 3 consecutive days at the same dose the expected cumulation was compensated for by an accelerated elimination of the substance, which is probably due to enzyme induction.     

HPLC-FLD测定多种植物提取物中苯并(a)芘的含量

目的建立测定植物提取物中苯并(a)芘含量的方法。方法采用HPLC-FLD法,用Agilent Li Chrospher PAH色谱柱(250 mm×4.6 mm),流动相为乙腈,检测器为荧光检测器(λex=365 nm,λem=470 nm),流速为1.0 ml.min-1,柱温30℃,进样量10μl。结果线性范围为1~25 ng.ml-1(r=0.9995);平均回收率为97.67%(RSD=9.85%)。结论所建方法重复性好、精密度高、能准确快速地测定植物提取物中苯并(a)芘的含量。     

Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on benzo(a)pyrene hydroxylase activity in embryos of the Japanese medaka (Oryzias latipes)

When Japanese medaka embryos were exposed to 12 ng/l 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) beginning on the day of fertilization (day 0), benzo(a)pyrene hydroxylase (B(a)PH) activity was induced in the whole embryo 105000g fraction by day 5 of development, which coincided with liver development. The induction of B(a)PH activity also coincided with the appearance of 2,3,7,8-TCDD induced hemorrhagic and edematous lesions. B(A)PH induction only occurred in embryos exposed to toxic concentrations (greater than 10 ng/l) of 2,3,7,8-TCDD. B(a)PH induction also occurred in embryos after exposure to 10 ng/l 2,3,7,8-tetrachlorodibenzofuran (TCDF) and 50 g/l 1,2,7,8-TCDD. Both 2,3,7,8-TCDF and 1,2,7,8-TCDD are toxic to Japanese medaka embryos at concentrations that resulted in the induction of B(a)PH activity. B(a)PH activity was not induced by the non-toxic congener 1,3,6,8-TCDD at concentrations as high as 50 g/l. The structure activity relationship for B(a)PH induction in Japanese medaka embryos was similar to that which is observed in other species and biological systems, suggesting that the biological activities of these compounds may also be mediated through the putativeAh receptor in these fish embryos. At 50 g/l, -naphthoflavone (BNF) induced B(a)PH activity in Japanese medaka embryos to similar levels as 2,3,7,8-TCDD did at toxic concentrations. However, at 50 g/l, BNF was not toxic to Japanese medaka embryos. Therefore, the induction of B(a)PH activity probably did not directly result in the toxicity observed in these fish embryos after exposure to 2,3,7,8-TCDD.Presented in part at the 28th Annual Meeting of the Society of Toxicology, Atlanta, GA, 1989.     

Evaluation of the precision-cut liver and lung slice systems for the study of induction of CYP1, epoxide hydrolase and glutathione S-transferase activities

The principal objective was to ascertain whether precision-cut tissue slices can be used to evaluate the potential of chemicals to induce CYP1, epoxide hydrolase and glutathione S-transferase activities, all being important enzymes involved in the metabolism of polycyclic aromatic hydrocarbons. Precision-cut rat liver and lung slices were incubated with a range of benzo[a]pyrene concentrations for various time periods. A rise in the O-deethylation of ethoxyresorufin was seen in both liver and lung slices exposed to benzo[a]pyrene, which was accompanied by increased CYP1A apoprotein levels. Pulmonary CYP1B1 apoprotein levels and hepatic mRNA levels were similarly enhanced. Elevated epoxide hydrolase and glutathione S-transferase activities were also observed in liver slices following incubation for 24h; similarly, a rise in apoprotein levels of both enzymes was evident, peak levels occurring at the same time point. When mRNA levels were monitored, a rise in the levels of both enzymes was seen as early as 4h after incubation, but maximum levels were attained at 24 h. In lung slices, induction of epoxide hydrolase by benzo[a]pyrene was observed after a 24-h incubation, and at a concentration of 1 microM; a rise in apoprotein levels was seen at this time point. Glutathione S-transferase activity was not inducible in lung slices by benzo[a]pyrene but a modest increase was observed in hepatic slices. Collectively, these studies confirmed CYP1A induction in rat liver slices and established that CYP1B1 expression, and epoxide hydrolase and glutathione S-transferase activities are inducible in precision-cut tissue slices.