Induction of hepatocellular carcinoma in rat liver by initial treatment with benzo(a)pyrene after partial hepatectomy and promotion by phenobarbital

The hepatocarcinogenicity of benzo(a)pyrene(BP) in the rat was examined. Rats were treated with BP after partial hepatectomy and then kept on a diet containing phenobarbital (PB) as a promoter. Tumors including a hepatocellular carcinoma developed in the rat liver by week 52.     

Induction of DNA damage by menadione (2-methyl-1,4-naphthoquinone) in primary cultures of rat hepatocytes

The cytotoxicity of menadione (2-methyl-1,4-naphthoquinone) had been investigated using primary cultures of rat hepatocytes. Menadione was found to induce DNA strand breaks which were actively repaired by the cells. Dicoumarol, an inhibitor of DT diaphorase, did not potentiate menadione-induced DNA strand breaks. Neither had metyrapone, an inhibitor of cytochrome P-450 dependent monooxygenases, any effect on the extent of DNA damage. Covalent binding of menadione metabolite(s) to DNA was detected in the cultured hepatocytes and, in addition, hepatic microsomes were also found to metabolize menadione to DNA-binding products. The extent of binding of menadione to DNA in vitro, was markedly decreased by inclusion of the hepatic cytosol fraction, or reduced glutathione, in the incubations. In the presence of dicoumarol, menadione was also found to induce cell membrane damage. It also caused a rapid loss in cellular glutathione which was augmented by the presence of dicoumarol. The results suggest that both the cell membrane damage and DNA damage induced by menadione are mediated by one-electron reduction of the quinone to free radical intermediate(s). DT diaphorase appears to protect the cell from membrane damage, whereas reduced glutathione may have an important role in the prevention of DNA damage.     

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.     

Chromosome aberrations induced by benzo(a)pyrene in a feeder cell-mediated assay

Studies were made on chromosome aberrations induced by benzo(a)pyrene (Bp) in V79 cells in the presence or absence of feeder cells. In the presence of feeder cells, chromosome aberrations at Bp concentrations of 1.0–20.0 μg/ml depended on feeder cell density. The highest incidences of chromosome aberrations (aberrant cells) and of aberrant chromosomes per 100 metaphase cells were 24.0% and 38.0%, respectively, and were obtained at 20.0 μg/ml Bp in the presence of 2.0 · 10⁶ feeder cells/60-mm plastic dish. In the absence of feeder cells, chromosome aberrations were not induced; the incidences of aberrant cells and chromosomes on treatment with Bp in the absence of a feeder layer were 3.0–5.0 and 3.0–6.0%, respectively, while the spontaneous rates (of both) were 5.0%.     

The effects of harman and norharman on the metabolism of benzo(a)pyrene in isolated perfused rat lung and in rat lung microsomes.

The effects of harman and norharman, nitrogen-containing pyrolysis products of amino acids present in cigarette smoke, on the metabolism of benzo(a)pyrene in rat lung microsomes in vitro and in isolated perfused rat lung were studied. In rat lung microsomes, both harman and norharman inhibited the metabolism of benzo(a)pyrene (BP) to dihydrodiols, phenols and quinones at concentrations over approximately 0.05 mM. The formation of BP-7, 8-dihydrodiol and BP-9, 10-dihydrodiol was inhibited more than that of BP-4, 5-dihydrodiol. No appreciable differences in inhibition were seen between microsomes from control or 3-methylcholanthrene-pretreated rats. In isolated perfused rat lung, 1 mM of harman in the perfusion fluid inhibited the formation of ethyl acetate-soluble metabolites of benzo(a)pyrene except BP-9, 10-dihydrodiol, and inhibited the total covalent binding of benzo(a)pyrene metabolites to lung tissue macromolecules. 0.03 mM of harman seemed to increase other metabolites than BP-7,8-dihydrodiol without changing the total covalent binding. These results suggest that at most concentrations both β-carboline derivatives, harman and norharman, inhibit benzo(a)pyrene metabolism and covalent binding both in lung microsomes in vitro and in isolated perfused rat lung.     

A toxicokinetic study to elucidate 3‐hydroxybenzo(a)pyrene atypical urinary excretion profile following intravenous injection of benzo(a)pyrene in rats

The toxicokinetics of benzo(a)pyrene (BaP) and 3‐hydroxybenzo(a)pyrene (3‐OHBaP) were assessed in 36 male Sprague–Dawley rats injected intravenously with 40 µmol kg¹ of BaP to explain the reported atypical urinary excretion profile of 3‐OHBaP. Blood, liver, kidney, lung, adipose tissue, skin, urine and feces were collected at t = 2, 4, 8, 16, 24, 33, 48, 72 h post‐dosing. BaP and 3‐OHBaP were measured by high‐performance liquid chromatography/fluorescence. A biexponential elimination of BaP was observed in blood, liver, skin and kidney (t_½ of 4.2–6.1 h and 12.3–14.9 h for initial and terminal phases, respectively), while a monoexponential elimination was found in adipose tissue and lung (t_½ of 31.2 and 31.5 h, respectively). A biexponential elimination of 3‐OHBaP was apparent in blood, liver and skin (t_½ of 7.3–11.7 h and 15.6–17.8 h for initial and terminal phases, respectively), contrary to adipose tissue, lung and kidney. In adipose tissue and lung, a monophasic elimination of 3‐OHBaP was observed (t_½ of 27.0 h and 24.1 h, respectively). In kidney, 3‐OHBaP kinetics showed a distinct pattern with an initial buildup during the first 8 h post‐dosing followed by a gradual elimination (t_½ of 15.6 h). In the 72‐h post‐treatment, 0.21 ± 0.09% (mean ± SD) of dose was excreted as 3‐OHBaP in urine and 12.9 ± 1.0% in feces while total BaP in feces represented 0.40 ± 0.16% of dose. This study allowed the identification of the kidney as a retention compartment governing 3‐OHBaP atypical urinary excretion. Copyright © 2010 John Wiley & Sons, Ltd.     

Induction of benzo[a]pyrene metabolism by 2,3,7,8-tetrachlorodibenzo-p-dioxin in primary cultures of adult rat hepatocytes

Primary hepatocyte cultures prepared from adult male Sprague-Dawley rats were extremely sensitive to induction of benzo[a]pyrene (BaP) metabolism by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), added to the cells in culture. A 48-hr exposure to 1 pmol TCDD/10⁶ cells resulted in a 4-fold induction of BaP metabolism. Significant induction was produced by a 48-hr exposure to a dose as low as 0.01 pmol (3 pg) TCDD/10⁶ cells. Cells exposed to TCDD from 7 to 55 hr in culture and then assayed for BaP metabolism responded to much lower doses of TCDD than did cells assayed at 36 hr in culture after being exposed to TCDD from 7 to 36 hr in culture. The doses of TCDD required for half-maximal induction of BaP metabolism were 24.8 and 164 fmol TCDD/10⁶ cells for cells assayed at 55 and 36 hr, respectively. Hepatocytes treated with TCDD starting at later times in culture showed a much more rapid time course of induction than did cells treated at earlier times in culture. A 2.6-fold induction occurred in cells treated from 49 to 55 hr in culture, compared to a 1.2-fold induction from 31 to 37 hr in culture. In contrast to cells exposed to TCDD from 7 to 55 hr in culture, cells exposed from 49 to 55 hr in culture were nearly as sensitive to inhibition of BaP metabolism by SKF 525-A as were uninduced control cells. An apparent derepression of the induction of BaP metabolism is occurring in primary hepatocyte cultures, resulting in an increase in the intitial rate of induction by TCDD and a decrease in the dose of TCDD required to obtain the half-maximum response.     

Activation of dioxin response element (DRE)-associated genes by benzo(a)pyrene 3,6-quinone and benzo(a)pyrene 1,6-quinone in MCF-10A human mammary epithelial cells

Benzo(a)pyrene (BaP) is a known human carcinogen and a suspected breast cancer complete carcinogen. BaP is metabolized by several metabolic pathways, some having bioactivation and others detoxification properties. BaP-quinones (BPQs) are formed via cytochrome P450 and peroxidase dependent pathways. Previous studies by our laboratory have shown that BPQs have significant growth promoting and anti-apoptotic activities in human MCF-10A mammary epithelial cells examined in vitro. Previous results suggest that BPQs act via redox-cycling and oxidative stress. However, because two specific BPQs (1,6-BPQ and 3,6-BPQ) differed in their ability to produce reactive oxygen species (ROS) and yet both had strong proliferative and EGF receptor activating activity, we utilized mRNA expression arrays and qRT-PCR to determine potential pathways and mechanisms of gene activation. The results of the present studies demonstrated that 1,6-BPQ and 3,6-BPQ activate dioxin response elements (DRE, also known as xenobiotic response elements, XRE) and anti-oxidant response elements (ARE, also known as electrophile response elements, EpRE). 3,6-BPQ had greater DRE activity than 1,6-BPQ, whereas the opposite was true for the activation of ARE. Both 3,6-BPQ and 1,6-BPQ induced oxidative stress-associated genes (HMOX1, GCLC, GCLM, and SLC7A11), phase 2 enzyme genes (NQO1, NQO2, ALDH3A1), PAH metabolizing genes (CYP1B1, EPHX1, AKR1C1), and certain EGF receptor-associated genes (EGFR, IER3, ING1, SQSTM1 and TRIM16). The results of these studies demonstrate that BPQs activate numerous pathways in human mammary epithelial cells associated with increased cell growth and survival that may play important roles in tumor promotion.     

小鼠苯并(a)芘的急性免疫毒性

<正> 苯并(a)芘(B(a)P),是煤焦油、煤烟及其它燃料不完全燃烧的产物,不仅污染了人类的生产、生活环境,给人类健康带来严重危害,而且具有致癌作用。关于B(a)P ip的免疫毒性,国内外未见报道。本文观察了B(a)P对小鼠体液免疫、细胞免疫及巨噬细胞功能的影响。 材料与方法 LACA佰♀健康小鼠,8～10周龄,体重22～25g,由北京医科大学实验动物部提供。实验分200,100,50 mg/kg三组,同时设溶剂对照组,染毒途径为一次ip。B(a)P,Sigma和Fluka公司生产。溶于玉米油或橄榄油,在磁力搅拌器上避光搅拌4～6h,使     

Induction of fibroblast growth factor-9 and interleukin-1alpha gene expression by motorcycle exhaust particulate extracts and benzo(a)pyrene in human lung adenocarcinoma cells.

Motorcycle exhaust particulates (MEP) contain carcinogenic polycyclic aromatic hydrocarbons including benzo(a)pyrene. This study has determined the ability of MEP to alter the expression of select genes from drug metabolism, cytokine, oncogene, tumor suppressor, and estrogen signaling families of human lung adenocarcinoma CL5 cells. cDNA microarray analyses and confirmation studies were performed using CL5 cells treated with 100 microg/ml MEP extract for 6 h. The results showed that MEP increased the mRNA levels of metabolic enzymes CYP1A1 and CYP1B1, proinflammatory cytokines interleukin (IL)-1alpha, IL-6, and IL-11, fibroblast growth factor (FGF)-6 and FGF-9, vascular endothelial growth factor (VEGF)-D, oncogene fra-1, and tumor suppressor p21. In contrast, MEP decreased tumor suppressor Rb mRNA in CL5 lung epithelial cells. Treatment with 10 microM benzo(a)pyrene for 6 h altered gene expression profiles, in a manner similar to those by MEP. Induction of IL-1alpha, IL-6, IL-11, and FGF-9 mRNA by MEP and benzo(a)pyrene was concentration and time dependent. Cotreatment with 2 mM N-acetylcysteine blocked the MEP- and benzo(a)pyrene-mediated induction. Treatment with MEP or benzo(a)pyrene increased IL-6 and IL-11 releases to CL5 cell medium. Incubation of human lung fibroblast WI-38 with MEP- or benzo(a)pyrene-induced CL5 conditioned medium for 4 days stimulated cell growth of the fibroblasts. Inhalation exposure of rats to 1:10 diluted motorcycle exhaust 2 h daily for 4 weeks increased CYP1A1, FGF-9, and IL-1alpha mRNA in lung. This present study shows that MEP and benzo(a)pyrene can induce metabolic enzyme, inflammatory cytokine, and growth factor gene expression in CL5 cells and stimulate lung epithelium-fibroblast interaction.     

Protective effects of lemongrass essential oil against benzo(a)pyrene-induced oxidative stress and DNA damage in human embryonic lung fibroblast cells

Benzo(a)pyrene (BaP) was a well-known environmental pollutant, numerous studies had implicated BaP as a causative agent in human cancer, particularly lung cancer. The lemongrass essential oil (LEO) possessed various pharmacological activities, especially the anti-oxidative stress and cancer prevention. In the current study, human embryonic lung fibroblast (HELF) cells were treated with 25?mM BaP in the absence or presence of 0.5%, 1% or 2.5% LEO and the cell viability and levels of oxidative stress (OS) and DNA damage in the cells were then measured. Nineteen chemical constituents were identified in LEO, with citral being the main component, representing about 68.78%. LEO was able to protect the HELF cells against BaP-induced loss in cell viability, achieving a maximum of 95.58% cell viability at the 0.5% concentration. Treatment of HELF cells with BaP alone significantly increased the level of Malondialdehyde (MDA) and decreased superoxide dismutase (SOD) and catalase (CAT). However, these effects were suppressed when the cells were also treated with LEO, leading to enhanced levels of SOD and CAT activities (2.9- and 2-fold, respectively, compared with BaP treatment only) and reduced the level of MDA in the cells (43% reduction in malondialdehyde level). At the same time, LEO also reduced the level of DNA damage, as shown by a reduced level of 8-hydroxy-deoxyguanosine (8-OHdG). Taken together, the results showed that LEO offered protection against BaP-induced OS and DNA damage, suggesting that LEO could be a promising agent for lung cancer chemoprevention.     

Molecular cloning of CYP1A gene and its expression by benzo(a)pyrene from goldfish (Carassius auratus)

We cloned and sequenced the cytochrome P450 1A (CYP1A) gene from goldfish (Carassius auratus). It has a 1581 bp open reading frame that encodes a 526 amino acid protein with a theoretical molecular weight of 59.02 kDa. The CYP1A amino acid sequence clusters in a monophyletic group with other fish CYP1As, and more closely related to zebrafish CYP1A (91% identity) than to other fish CYP1As. Exposure to benzo(a)pyrene (BaP) by intraperitoneal injection increased biliary BaP metabolites and liver CYP1A gene expression. BaP exposure also increased CYP1A gene expression in extrahepatic organs, including intestine, and gill, which are sensitive to aqueous and dietary exposure to Arylhydrocarbon receptor (AhR) agonists. Therefore, goldfish CYP1A identified in this study offers basic information for further research related to biomarker use of CYP1A of goldfish.     

Protection against toxic redox cycles between benzo(a)pyrene-3,6-quinone and its quinol by 3-methylcholanthrene-inducible formation of the quinol mono- and diglucuronide

Cytotoxic effects of quinones are thought to be mediated by redox cycles between quinones and quinols whereby reactive oxygen species are generated. The role of glucuronidation in preventing these toxic redox cycles was investigated by using benzo(a)pyrene-3,6-quinone and isolated rat hepatocytes or Reuber hepatoma cells (H4IIE). Inhibition of quinol glucuronidation by salicylamide enhanced quinone-dependent oxygen uptake and cytotoxicity. Conjugation of benzo(a)pyrene-3,6-quinol was shown to proceed via the 6-monoglucuronide to the diglucuronide. Diglucuronide formation was low in hepatocytes from untreated controls and phenobarbital-treated rats. However, it was highly stimulated (26-fold) in hepatocytes from 3-methylcholanthrene-treated rats and was also high in Reuber hepatoma cells. Kinetic analysis with liver microsomes indicated that 3-methylcholanthrene-stimulated glucuronidation was due to an increased Vmax of UDP-glucuronosyltransferase which was enhanced 10- and 40-fold or mono- and diglucuronide formation, respectively. These findings suggest that the investigation of quinol glucuronidation (in particular the formation of benzo(a)pyrene-3,6-quinol diglucuronide) is a most useful probe for the 3-methylcholanthrene-inducible isoenzyme(s) of UDP-glucuronosyltransferase. Moreover, this isoenzyme may be particularly suited to protect against toxic redox cycles between benzo(a)pyrene quinones and quinols.     

Phytol ameliorated benzo(a)pyrene induced lung carcinogenesis in Swiss albino mice via inhibition of oxidative stress and apoptosis

In the present study, the modulatory effect of phytol against benzo(a)pyrene [B(a)P] induced lung carcinogenesis was investigated in Swiss albino mice. During the experimental period, phytol treatment showed no adverse toxic effect and mortality to the experimental animals. Lung tumor was observed in B(a)P treated group and also in animals post‐treated with low concentration (50 mg/kg) of phytol. No neoplastic changes were observed in the lung tissue of the animals treated with the maximum dose of phytol (100 mg/kg). An elevated level of antioxidant enzymes combined with macromolecular damage (lipid peroxidation, protein carbonyl content) was observed upon B(a)P treatment whereas, phytol restored the level of antioxidant enzymes which were comparable to the vehicle control group. Moreover, administration of B(a)P induced apoptosis, as observed by the highest expression of Bax, caspase‐3, and caspase‐9 proteins in lung tissue of B(a)P alone treated animals. However, phytol treatment reduced the expression of Bax, caspase‐3, and caspase‐9 protein and maintained the constant expression of anti‐apoptotic protein Bcl‐2. These observations positively reveal that phytol regulates the antioxidant enzymes and thereby protects the cells against B(a)P induced carcinogenesis without showing any adverse toxic effect to the animals.     

Aryl hydrocarbon (benzo[a]pyrene) hydroxylases in liver from rats of different age, sex and nutritional status. Distinction of two types by 7,8-benzoflavone.

Two types of aryl hydrocarbon (benzo[a]pyrene) hydroxylase have been distinguished in liver from rats of different sex and age by their sensitivity to the synthetic flavonoid, 7,8-benzoflavone. One type, which is stimulated by the 7,8-benzoflavone, is found in newborn rats and predominates in the liver of adult male rats. This type is inducible by phenobarbital. A second type, which is inhibited by 7,8-benzoflavone, comprises a larger fraction in the liver of adult female rats and is inducible by polycyclic hydrocarbons in immature and mature animals of either sex. The presence of this form in adult female liver is also indicated by the kinetics of the hydroxylase reaction. Removal of solid food for 18 hr not only decreases hepatic aryl hydrocarbon hydroxylase activity in female rats, but also lowers the degree of inhibition by 7,8-benzoflavone. Kinetic data suggest that at low concentrations 7,8-benzoflavone acts as a competitive inhibitor but at higher concentrations inhibits the hydroxylation reaction by a more complex mechanism.     

Effect of benzo(a)pyrene exposure on fluoranthene metabolism by mouse adipose tissue microsomes

The present study has been undertaken to examine whether exposure to benzo(a)pyrene (BaP), a polycyclic aromatic hydrocarbon (PAH) compound, influences the metabolism of fluoranthene (FLA), another PAH compound. Microsomes were isolated from the adipose tissue of mice that received 50?μg/kg BaP and incubated with FLA (3?µM) alone; FLA in combination with BaP at equimolar concentrations, and a control group that received nothing. Post-incubation, samples were extracted with ethyl acetate and analyzed for FLA metabolites by reverse-phase HPLC with fluorescence detection. The rate of FLA metabolism (pmol of metabolite/min/mg protein) was increased when microsomes from BaP-treated mice were exposed to FLA alone and FLA in combination with BaP, compared to controls. On the other hand, the difference in FLA metabolic rate between microsomes that were exposed to FLA + BaP was higher than the ones that received FLA. The microsomes from BaP-pre-treated mice produced a considerably higher proportion of FLA 2, 3-diol, and 2, 3 D FLA when microsomes were incubated with FLA. There were no differences in the FLA metabolite types formed when BaP-pre-treated mice were co-incubated with BaP and FLA than with FLA alone. The enhanced biotransformation of FLA as a result of prior and concomitant exposure to BaP may have implications for assessment of risks arising from human exposure to PAH mixtures.     

Sulfur dioxide and benzo(a)pyrene modulates CYP1A and tumor‐related gene expression in rat liver

Sulfur dioxide (SO₂) and benzo(a)pyrene (B(a)P) are common industrial and environmental contaminants. However, few data are available on the effects of SO₂ on proto‐oncogenes and tumor suppressor genes, as well as the interactions between SO₂ and other xenobiotics regulating proto‐oncogenes or tumor suppressor genes expression. To investigate the interactions between SO₂ and B(a)P, male Wistar rats were exposed to intratracheally instilled with B(a)P or SO₂ inhalation alone or together. We detected mRNA expression of CYP1A1 and 1A2, 7‐ethoxyresorufin O‐deethylase (EROD), and methoxyresorufin O‐demethylase (MROD) activities in livers. The mRNA and protein levels of several cancer‐related genes were analyzed in livers by real‐time RT‐PCR and Western blot, respectively. The EROD/MROD activities and CYP1A1/2 expression were down‐regulated by SO₂ but up‐regulated by B(a)P alone. Exposure of SO₂ alone induced c‐fos, c‐jun, c‐myc, H‐ras, and p53 expression, and depressed p16 and Rb expression in livers. The effects of B(a)P on the above gene were similar to SO₂ except c‐fos expression. Furthermore, SO₂ + B(a)P exposure increased the expression of c‐fos, c‐jun, c‐myc, and p53, and decreased p16 and Rb expression in livers compared with exposed to SO₂ or B(a)P alone. However, no synergistic effects were observed on H‐ras and CYP1A1/2 after SO₂ + B(a)P exposure. Our findings indicate that multiple cell cycle regulatory proteins play key roles in the toxicity of SO₂ and B(a)P in livers. It might involve the activation of c‐fos, c‐jun, c‐myc, and p53. And p16‐Rb pathway might also participate in the progress. Although the gene products we studied are classed as oncogenes and tumor suppressor genes, their functions actually relate to more general processes of control of cell proliferation, survival, and/or apoptosis. © 2009 Wiley Periodicals, Inc. Environ Toxicol, 2010.