Preliminary evidence for the metabolism of benzo(a) pyrene by Plantago lanceolata

Polycyclic aromatic hydrocarbons (PAHs) have long been recognised as potential carcinogens in animals in which biotransformation into reactive metabolites can lead to DNA damage. In animals PAHs metabolism mainly occurs in hepatic microsomes and is associated with the cytochrome p-450 mediated mixed functional oxidase (MFO) system. PAH metabolism in plants has been shown to occur via a similar enzyme system, but has received relatively little attention. This study is looking at how the plant species Plantago lanceolata metabolises benzo(a)pyrene (B(a)P), which is one of the PAHs whose metabolism has been studied extensively in animals. The aim of the work is to establish firstly that the B(a)P is taken up and secondly that it is biotransformed by the plant to products possibly similar to those found in animals. This work is achieved by using C-14-B(a)P along with whole body autoradiography, scintillation analysis and chromatography techniques to locate the B(a)P and its metabolites.     

Gender differences in the metabolism of benzene,toluene and trichloroethylene in rat with special reference to certain biochemical parameters

Metabolites viz. phenol, hippuric acid and total trichloro compounds of benzene, toluene and trichloroethylene respectively were estimated in the urine samples of male and female rats after exposure for a period of 30 days. The results exhibited higher metabolism in female rats than the male rats. Their metabolism might be regulated by cytochrome P450 isozymes in a gender specific manner. However, sex differences in the activity of glutathione-S-transferases of the liver have also been found to determine their toxicity. Results have been discussed with quantitative profiles of other enzymes established in the liver of male and female rats.     

Cytochrome P450 1A activity in liver and fixed wavelength fluorescence detection of polycyclic aromatic hydrocarbons in the bile of tongue-fish (Cynoglossus acrolepidotus, Bleeker) in relation to petroleum hydrocarbons in the eastern Gulf of Thailand.

This investigation was conducted in an area of oil spill along the east coast of Thailand to examine the relations among cytochrome P450 1A activity in liver and PAHs in the bile of the tonguefish and petroleum hydrocarbons in the sediments. PAH sediment concentrations in the reference and oil spill areas were 5.03 +/- 0.42 and 0.21 +/- 0.043 microg(-1) dry weight respectively Cytochrome activity in fish liver from oil spill area was 45.40 +/- 3.50 pmoles/ min/mg protein, almost threefold higher than that from the reference sites. Flourescense detection in bile metabolites at the oil spill area, 69.8 +/- 9.9 flourescense unit was significantly higher than that at the reference sites, 22.9 +/- 5.5 and 22.2 +/- 3.5 fluorescence unit. A strong correlation was found among cytochrome P450 1A activity in liver, PAH of bile metabolites and petroleum hydrocarbons. Both cytochrome and bile metabolites activity decreased seaward varying to the distance from the oil polluted area. We concluded that both detections in tonguefish can be regarded as a complementary biomarkers for the exposure of PAHs in tropical marine environments.     

Isolation and sequence of cDNA encoding a 3-methylcholanthrene-inducible cytochrome P450 from wild red sea bream,Pagrus major

We have isolated a cDNA clone of mRNA for the cytochrome P450 from a 3-methylcholanthrene (MC)-treated red sea bream,Pagrus major, using a cDNA fragment for rat P4501A2 as a probe. The cloned cDNA is ca. 1.8 kb long and contains an open reading frame of 1545 nucleotides for polypeptides of 515 amino acids. The deduced N-terminal amino acid sequence of the cDNA is very similar to that for purified cytochrome P450 protein from the marine fish scup, which was reported previously (Klotz et al. 1983). A conserved amino acid sequence containing a putative heme-binding cysteine is present in the equivalent position proximate to the C-terminus of the molecules. The deduced amino acid sequence shows more than 50% positional identity with known members of the mammalian aromatic hydrocarbon-inducible P450 family. RNA blot analysis indicates that P450 mRNA (s) is expressed in the liver, kidney, gill and gut of the MC-treatedP. major.     

Influence of testosterone on phase-II enzymes in liver and kidney of benzene-treated rats

Testosterone manifests some protective effects against benzene-induced toxicity in rats. However, mechanism of protection remains to be established. Data showed that testosterone modulates conjugation of reactive metabolites of benzene by influencing phase-II enzymes viz. glutathione-S-transferase, glutathione peroxidase, and catalase in both liver and kidney. These observations are supported by the opposite results obtained in castrated rats. It is postulated that testosterone decreases the formation of reactive oxygen species resulting into an increase in phase-II enzymes. Enhanced activity of these antioxidant enzymes is responsible for DNA strand repair as demonstrated by short comet tail length in liver and kidney of benzene and testosterone treated rats. Castration alters benzene pharmacokinetics by influencing these enzymes, a response which may be abolished by testosterone supplementation.     

恩诺沙星在鲫鱼肝微粒体中的代谢及代谢关键酶的确定

抗生素因具有抗菌谱广、杀菌性强等特点使其被广泛应用于人类医药、畜牧业、农业和水产养殖业。其进入水生生物体内后,会在药物代谢酶的作用下发生代谢转化,产生生态毒性。本研究采用鲫鱼肝微粒体体外孵育法,探究恩诺沙星细胞色素P450酶作用下的代谢转化过程,并通过代谢抑制实验确定关键的代谢酶。实验结果表明,恩诺沙星体外代谢过程符合一级动力学方程,当恩诺沙星暴露浓度为1 mg·L-1时,其在肝微粒体中的消除速率常数k最大为0.00303 min-1,半衰期t1/2最短为228.8 min,应用HPLC-MS/MS技术,检测到了恩诺沙星脱乙基产物和羟基化产物;代谢抑制实验结果表明,CYP3A4在恩诺沙星代谢过程中起主要作用,是恩诺沙星代谢的关键酶。本研究结果为深入了解恩诺沙星在水生生物体内的代谢转化及其生态风险提供了基础数据。     

Benzo[a]pyrene oxidation and microsomal enzyme activity in the mussel (Mytilus edulis) and other bivalve mollusc species from the Western North Atlantic

Analysis of subcellular fractions revealed a complement of microsomal electron transport components including reductases and heme proteins in several organs of the three bivalve species Mytilus edulis, Macrocallista maculata and Area zebra. Dithionite difference spectroscopy of CO-treated microsomes yielded spectra typical of cytochrome P-450 in digestive gland and gill, with absorption maxima at 450 nm. A time-dependent reduction of cytochrome P-450 was also observed. The levels of these components and rates of microsomal benzo[a]pyrene (BP) metabolism were highest in the digestive gland, and were very similar between species. In M. edulis there was a suggested seasonal variation in BP metabolism but no population differences in this activity or in levels of other components. Digestive gland microsomal metabolites of BP identified by HPLC retention and UV spectroscopy included BP-1.6-quinone, BP-3,6-quinone and BP-6,12-quinone, which comprised 65% of the total metabolites, and dihydrodiols and phenols, the latter products consistent with cytochrome P-450 monooxygenation and expoxide hydrolase function. However, the inconsistent dependence of BP metabolism on NADPH, and inconsistent inhibition by CO suggest that catalyst(s) additional to cytochrome P-450 may be acting in BP metabolism. Based on these results and the prominent quinone formation, we speculate that peroxidative mechanism(s) may be involved. The role of peroxidative as well as well as monooxygenase reactions in the in-vivo disposition and effects of foreign chemicals in bivalves, and also the major function of cytochrome P-450 in these bivalves, remain to be established.Some of these results have appeared in preliminary form; J. J. Stegeman, Sea Grant Annual Report, Words Hole Oceanographic Institution, p 15, 1981     

苯急性暴露对Sprague-Dawley大鼠全基因组DNA甲基化的影响

现有文献表明,DNA甲基化异常与肿瘤的发生密切相关,其中全基因组DNA甲基化水平的改变已经被认为是癌症发生的生物标志物;同时,大量遗传毒理学实验证明苯可以引起DNA突变和断裂,然而苯暴露引起全基因组DNA甲基化异常的现象,目前鲜有文献报道。为了揭示苯引起全基因组DNA甲基化变化的致毒机制,本实验中,Sprague-Dawley（SD）雄性大鼠经口灌胃急性暴露于以500 mg.kg-1（以体质量计）的苯中,在暴露6、12、24、30 h后采集SD雄性大鼠体内血液、肝脏、肾脏和肺,利用高效液相色谱分析方法检测全基因组DNA甲基化水平。结果表明,SD雄性大鼠血液和肝脏的全基因组DNA甲基化水平显著下降,而在肾脏和肺中没有显著地变化,表现出组织特异性。本实验率先报道了苯的暴露可以引起全基因组DNA甲基化水平的异常,从表观遗传学的角度解释了苯影响人体健康的机制。     

Organ differences in microsomes and cytosol metabolism of Aflatoxin B1 in piglets

In order to delineate the features of aflatoxin B1 (AFB1) metabolism in various organs of piglets, in vitro metabolism of AFB1 by microsomes and cytosol of the various piglet organs was studied. The AFB1 was converted efficiently to AFP1 by the kidney microsomes. A less efficient metabolism was noted from the AFB1 to AFQ1, AFM1, and aflatoxicol (AFL) in the various organs. The microsomal ability to form AFB1-DNA adduct was higher in liver when compared to the other organs. The cytosolic glutathione-S-transferase activity to convert AFB1-epoxide to AFB1-glutathione conjugate product was relatively higher in the liver and the small intestine. The reductase activity to convert AFB1-dialdehyde to AFB1-dialcohol was similar in all the organs. The results suggest that the variation in susceptibility to the aflatoxin among different organs is attributable mainly to the organ differences in cytochrome P450 activity to form AFB1-epoxide.     

Characterization of the liver cytochrome P450 in the marine fish Mugil cephalus and effects of some hydrocarbons on the mixed-function oxidase components

The marine fish Mugil cephalus was caught in the Bay of Marseilles (France) in 1985 and the liver microsomal cytochrome P₄₅₀ was identified at a concentration of about 0.230 nmol mg^-1 protein. Optimal assay conditions were established to measure two mixed-function cytochrome P₄₅₀-dependent oxidases: aniline hydroxylase and p-nitroanisole O-demethylase. Induction of cytochrome P₄₅₀ occurred after exposure of mullet to an aromatic hydrocarbon and a water-soluble petroleum fraction. The liver cytochrome P₄₅₀ was solubilized and purified. The monomer molecular weight of partially purified cytochrome P₄₅₀, determined by sodium dodecyl-sulfate-gel electrophoresis, was 51 000.     

Distribution and binding pattern of benzo(a)pyrene in rat liver,lung and kidney constituents after oral administration

Orally administered ³H‐benzo(a)pyrene (BP) was persistent in protein fraction of liver, lung and kidney. The radioactivity in this fraction increased with time after administration and accounted for about 50%, 40% and 65% of total radioactivity in liver, lung and kidney, respectively at 48 hr. The BP metabolites binding proteins were located in cytosol and had molecular weights of 40,000–60,000 and 80,000–100,000 as determined by gel filtration and polyacrylamide gel disc electrophoresis. In addition, at 48hr after administration, about 80% of radioactivity in high molecular weight protein fraction was found to be precipitated by trichloroacetic acid treatment.

These results suggest that BP metabolites might be transported by and are persistent in these protein fractions of liver, lung and kidney if the intake of BP is continuous. These proteins, therefore, appeared to be closely related to cell toxicity or mutagenicity in these organs as well as DNA.     

Stoffströme von Benzol unter besonderer Berücksichtung der Bundesrepublik Deutschland

In the Federal Republic of Germany, benzene is one of the most important basic materials for the chemical industry. Only a relatively small proportion of the pure benzene processed in the chemical industry is emitted into the environment (ca. 40t in 1991). But the substance is also a natural component of the crude oil in gasoline and is released during incomplete combustion or is formed out of other aromatic substances. The primary source of emissions, with more than 10,000 t/yr (approximately 85% of this from motor vehicles with Otto engines), is commercial motor vehicle transportation. Benzene concentrations in the environment are approximately <1 μg/m³ in rural areas, 20–30 μg/m³ near main roads (peak levels in highly urbanized regions with much traffic as high as approximately 100 μg/m³) and 7–15 μg/m³ in the vicinity of industrial polluters. It has not been possible to detect a specific trend over time during the last ten years. An increased exposure (approx. 350–27,000 μg/m³) is likely while filling the tank and within the motor vehicle (approx. 10–200 μg/m³) due to gasoline volatilization from pipes, etc. Compared to outdoor air, higher concentrations of benzene (approx, 2–11 μg/m³) are measured in the indoor air which contains additionally benzene from tobacco smoke, equipment, renovating work and heating. The primary exposure pathway of benzene in humans is inhalation. Apart from individuals with occupational exposure, smokers have the highest internal benzene burden. Measures undertaken during recent years to reduce the amount of emissions have been counteracted at least in part by the increase in motor vehicle traffic. Further measures to reduce the emissions or to change the transporttation policies must still be or have already been initiated.     

The effect of lead bioaccumulation on haem biosynthetic enzymes in fish

The bioaccumulations of lead in the liver and hepatic microsomes of fish after 1, 3, 7, 14, 28, and 45 days exposure were studied. In addition, the relationship between the bioaccumulated lead in both hepatic microsomes and the liver and their haem biosynthetic enzymes were studied. Lead toxicity was shown to result in a depression of the microsomal mixed function oxidase system, as assessed by a decrease in hepatic microsomal cytochrome P-450 and b5 content and by a decrease in the activity of the enzymes aniline hydroxylase and aminopyrine demethylase. Lead had a more marked effect on cytochrome P-450 than b5. The activity of the rate-limiting enzyme of haem biosynthesis, delta-aminolevulinic acid synthase, was inversely correlated with the microsomal cytochrome P-450 content. The activity of the heam biosynthetic enzymes delta-aminolevulinic acid dehydratase, coproporphyrinogen oxidase and ferrochelatase were decreased by increasing lead pretreatment. The activity of the haem catabolic enzyme, haem oxygenase, was increased by concentration and length of time to lead exposure.     

Effects of nonylphenol on the brain cytochrome P450 gene expression in F1 generation rats

In order to explore the effects of nonylphenol (NP) on brain cytochrome P450 gene expression in F1 generation rats microarray analysis techniques were used. mRNA were extracted from the brain of 2-day-old F1 generation rats whose F0 male generation were treated with NP, then reversely transcribed to cDNA and labeled with cy5 and cy3 for fluorescence. Subsequently, the cDNA probes were hybridized to the mouse 40S cDNA microarray; and fluorescent signals produced by cy5 and cy3 were scanned and analyzed. Sixteen identified genes were found to be expressed differently from control, including three cytochrome P450 genes, in which two were up-regulated and one down-regulated. Data suggest that NP affects the expression of some cytochrome P450 genes in rat brain when administered perinatally.     

Studies on cytochrome P450 in Mytilus galloprovincialis: induction by Na-phenobarbital and ability to biotransform xenobiotics

Mytilus galloprovincialis has a hepatopancreatic monooxygenase activity cytochrome P₄₅₀-dependent. The present paper studies the induction of this activity in the hepatopancreatic microsomes after treatment of M. galloprovincialis with Na-phenobarbital. Mussels were collected from the Gulf of La Spezia in 1985. We measured the increase in levels of cytochrome P₄₅₀, b₅, and P₄₂₀ and the 7-ethoxycoumarin deethylase activity. Furthermore, the fraction of mouse liver was replaced by the correspondent mussels' hepatopancreatic fraction in the yeast genotoxicity test to determine the ability of mussels to biotransform cyclophosphamide and styrene oxide. The results suggest that edible mussels are capable of detoxicating styrene oxide but not of bioactivating cyclophosphamide.     

Toxicological and ecological implications of biotransformation enzymes in the tropical teleost Chaetodon capistratus

Hepatic cytochromes P450 (phase I monooxygenases) and glutathione transferases (phase II conjugating enzymes) were investigated in Chaetodon capistratus (Linnaeus) collected in Florida and Belize in June and December 1991, respectively. These biotransformation enzymes play major roles in the detoxification of xenobiotics by converting lipophilic chemicals to more hydrophilic, readily excretable metabolites. Content of total microsomal P450 (0.501 to 0.821 nmol mg^-1 microsomal protein) and rates of NADPH-cytochrome c (P450) reductase (270.7 to 330.2 nmol min^-1 mg^-1 microsomal protein) and glutathione transferase (2.81 to 3.12 g min^-1 mg^-1 cytosolic protein) in these fish were greater than in most untreated fish species, i.e., fish that have not been exposed to PAHs (polycyclic aromatic hydrocarbons) or PCBs (polycyclic biphenyls). Ethoxyresorufin O-deethylase (EROD) rates (0.029 to 0.171 nmol min^-1 mg^-1) were also comparable to those in most untreated marine fish. Immunoblot analysis with monoclonal antibody (MAb) 1-12-3 to scup P450E (the EROD catalyst and a teleost representative of the PAH-inducible CYP1A gene subfamily) showed slight amounts of cross-reacting protein in C. capistratus liver microsomes. Hepatic CYP1A content and EROD activity did not differ significantly between fish collected in Florida and Belize, suggesting that the two sites differed little in contamination by CYP1A inducers. Immunochemical analyses with polyclonal antibodies to scup P450B (a teleost representative of the CYP2B subfamily) and human CYP3A4 cross-reacted strongly with C. capistratus hepatic proteins. The CYP2B and CYP3A subfamilies in mammals are believed to have partially evolved in response to toxic dietary allelochemicals. C. capistratus regularly feeds on terpenoid-rich gorgonian corals, suggesting that biotransformation enzymes may be involved in the metabolism of dietary allelochemicals as well as anthropogenic xenobiotics in this species.     

硫丹对草鱼Ⅰ相、Ⅱ相酶活性及DNA损伤的影响

研究了硫丹对草鱼肝脏Ⅰ相酶氨基比林-N-脱甲基酶（APND）和红霉素-N-脱甲基酶（ERND）、Ⅱ相酶谷胱甘肽-S-转移酶（GST）活性及DNA受损细胞彗星尾长（TL）和尾部DNA含量（%TDNA）的影响。试验共设置0.18、0.36和0.71μg.L-1 3个暴露浓度组和1个空白对照组,分别在试验24、72、120和168 h时取样测定各指标。结果表明,24 h时,0.36和0.71μg.L-1暴露组草鱼肝脏APND活性与对照组相比显著升高（P〈0.05或P〈0.01）,72 h时受到显著抑制（P〈0.01）;120 h后各暴露组APND活性与对照组相比均表现为受到显著抑制（P〈0.05或P〈0.01）。ERND活性总体表现为受诱导。GST活性总体呈先受诱导后受抑制的变化趋势;0.36和0.71μg.L-1暴露组GST活性均在72 h时达到最高值,之后随暴露时间的延长缓慢降低,并在168 h时表现为受抑制;0.18μg.L-1暴露组在120 h时达到最大值,之后降低,168 h时GST活性与对照组水平相当。经硫丹暴露后,草鱼肝脏细胞DNA明显受损,TL与%TDNA均随硫丹浓度的升高或暴露时间的延长而增加,且相关显著。硫丹可影响草鱼肝脏Ⅰ相、Ⅱ相代谢酶活性,并对肝细胞DNA造成遗传损伤。     

Mutagenicity of polyaromatic hydrocarbons by chemical models for cytochrome P450 in Ames assay

DNA damage is an important step in carcinogenesis. The Ames assay is a short-term screening of carcinogens that induce DNA damage. Most carcinogens require enzymatic activation through oxidation by cytochrome P450 (CYP450) in the presence of S9 mix. A combination of iron (Fe)(III) porphyrin and an oxidant is also able to oxidize compounds as an alternative metabolic pathway to CYP450. Previously it was reported that a chemical model containing a water-soluble 5,10,15,20-tetrakis(1-methylpyridinium4-yl)porphyrinatoiron(III) chloride (4-MPy) and tert-butyl hydroperoxide (t-BuOOH) activated aromatic amines and amides. In this study, a chemical model composed of an Fe porphyrin, water-insoluble 5,10,15,20-tetrakis(pentafluorophenyl)porphyrinatoiron(III) chloride (F₅P) or water-soluble 4-MPy was optimized with an oxidant – t-BuOOH, magnesium monoperoxyphthalate (MPPT), or iodosylbenzene (PhIO). Subsequently the mutagenicity of benzo[a]pyrene (B[a]P) and chrysene in Salmonella typhimurium TA strains was compared. B[a]P was activated by a combination of F₅P or 4-MPy plus MPPT or PhIO in S. typhimurium TA1538. The B[a]P-induced mutagenicity with F₅P plus oxidant was higher than 4-MPy plus oxidant. Mutagenicity of chrysene, a tetracyclic aromatic hydrocarbon, was not detected in the presence of F₅P/PhIO in S. typhimurium TA98, but was activated in the presence of F₅P/MPPT. The F₅P/MPPT activated other polyaromatic hydrocarbons (PAH) in the S. typhimurium TA98 assay including dibenz[a,c]anthracene, dibenz[a,h]anthracene, 3-methylcholanthrene, and benzo[a]anthracene. The results indicated that the F₅P/MPPT was the most efficient model for detecting PAH-induced mutagenicity in the Ames assay.     

Bromobenzene-induced hepatotoxicity in male rats: the protective effect of flaxseed

The metabolites of bromobenzene (BB) are hepatotoxic. The aim of this study was to determine the efficacy of different doses of flaxseed extract in alleviating BB-hepatotoxicity in male albino rats. Oxidative stress parameters, drug metabolizing enzymes, a pro-inflammatory marker, an apoptotic marker, and DNA fragmentation pattern were also assessed. Animals were divided into five groups treated by intragastric intubation as follows: control, BB-treated 460?mg?kg^?1?BW alone; three animal groups (III, IV, V) were treated concurrently with 460?mg?kg^?1 BB daily for 3 weeks and different doses of flaxseeds extract: 100, 200, or 300?mg?kg^?1?BW. Oral treatment of BB produced a significant decrease in activities of antioxidant enzymes superoxide dismutase and glutathione peroxidase and glutathione levels, while activities of glutathione reductase and drug-metabolizing enzymes; glutathione-S-transferases and cytochrome P450 were enhanced. BB-treatment resulted in enhanced production of nitric oxide and activation of COX-2 and caspase-3. Pre-treatment with different doses of flaxseeds extract prior and during BB-treatment protected liver against BB-induced hepatotoxicity. The lower dose of flaxseed extract (100?mg?kg^?1) was most effective one.     

有机氯农药硫丹的生殖毒性及其机制研究进展

硫丹作为一种广谱有机氯农药,在农业区域周边的土壤和水体中都存在较高残留,2011年被斯德哥尔摩公约列入持久性有机污染物(POPs)名单。硫丹已被证实对神经系统、心血管系统、肝、肾等具有毒性作用,关于其生殖毒性的研究很多,但潜在机制尚不完全清楚。本文总结了硫丹导致的不同动物的生殖毒性,并从生殖器官和生殖细胞损伤、氧化应激以及DNA损伤、生殖细胞周期阻滞及细胞凋亡等方面,对近年来硫丹生殖毒性及其作用机制研究进展进行综述,并对其中存在的问题进行讨论,以期有助于深入了解硫丹的毒性效应。