Liver Enzyme Induction and Cancer

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Liver Enzyme Induction and Cancer

Introduction

Research shows that certain PCBs tend to PROMOTE cancer, once the cancer has been started by another cause. PCBs stimulate the production of important liver enzymes, which activates key metabolic processes, which enhances the cancer-causing activity of other chemicals. (Certain chemicals cause cancer or mutations only after being metabolized into byproducts.) As a result, PCBs may increase the cancer risks of tobacco smoking, air pollutants, or contaminants found in our food and water, as discussed in the studies below.

A commercial mixture of PCBs, called Aroclor 1254, is used routinely by laboratories to induce liver enzymes in order to test the ability of other chemicals to cause mutations or cancer. Commercial labs currently sell pre-made enzyme mixtures induced by Aroclor 1254 in rat livers, for use in this medical research. (See In Vitro Technologies website.)

This combined toxicity effect would be extremely difficult to sort out in an epidemiological study of humans in the real world. Toxicologists usually adjust cancer statistics in PCB studies to rule out cancer cases from tobacco smoking or other known cancer risk factors, when, in fact, PCB enzyme induction might have contributed to some of these cancers.

The Liver Induction Studies

The following 29 studies demonstrate PCBs’ well-known ability to stimulate the production of liver enzymes and provide examples of the use of PCB-induced enzymes for the purpose of testing the toxicity of other chemicals. Though PCBs were not discussed in some of the abstracts, the TOXNET database noted that PCBs were used as the "inducer" in those studies. This is not a complete list of such research. For more examples, visit the TOXNET database, the source of these abstracts.

Study #1

PCBs are potent inducers of liver cytochrome P450 in various species
PCB commercial mix Aroclor 1254 induced ECOD enzyme activity in human liver cancer cells (HepG2)
Aroclor 1254 also induced EROD enzymes in human liver cancer cells
PCB 77 had no effect on human liver cancer cells

Polychlorinated biphenyls are potent inducers of hepatic cytochrome P450 in various species. Until now, no model based on cultured cells can be considered as a universal surrogate for in vivo metabolism. In this respect, cultured rat hepatocytes, quail hepatocytes, and human hepatoma (HepG2) cells were used to study the effects of 3,3',4,4'-tetrachlorobiphenyl (3,3',4,4'-TCB) and Aroclor 1254 on drug-metabolizing enzymes. The presence of dexamethasone in the culture medium allows the expression and the induction of several cytochrome P450 isoenzymes found in adult cells. Induction of ethoxycoumarin-(ECOD) and ethoxyresorufin-O-deethylase (EROD), activities were measured. Induced P450s were identified by immunoblotting and Northern blotting. Aroclor 1254 induced ECOD activity in all three cell types, but the effect was much stronger in fetal rat hepatocytes than in human or quail cells. Aroclor failed to induce EROD activity in quail cells, had a slight inducer effect in HepG2 cells, and a marked effect in rat hepatocytes. 3,3',4,4'-TCB had no effect in HepG2 cells but significantly increased EROD and ECOD activities, especially the latter, in rat and quail cells. On the immunoblots, specific antibodies revealed essentially CYP1A1 in fetal rat hepatocytes, CYP2B1/2 in quail hepatocytes and CYP3A1 in HepG2 cells. Analysis of Northern blots showed an hybridization with CYP1A1, 2B1 and 3A1 mRNA in fetal rat hepatocytes, CYP3A and 1A mRNA in HepG2 cells, and a form of CYP2 mRNA in fetal quail hepatocytes closely related to homolog rat CYP2E or CYP2C. In quail hepatocytes, induction did not increase proportionally with the concentration of inducer in the culture medium. Instead, the dose-response curves (for EROD activity especially) peaked sharply at 1 muM Aroclor 1254, an effect attributed to changes in membrane fluidity or lipid content. Our results highlight the advantage of using several types of cultured hepatocytes to investigate fundamental aspects of drug-metabolism-linked toxicity, the balance between xenobiotic bioactivation and detoxication being differently affected by PCBs in different animal species. (Dubois et al, 1996)

Study #2

enzyme induction by dioxin and dioxin-like PCB congeners (coplanars) infers they are present at levels that are potentially toxic, carcinogenic, or mutagenic to organisms

In this study we describe the use of a transgenic cell line for the identification of potentially toxic compounds in test solutions and environmental samples. The reporter gene system (RGS), derived from a human liver cancer cell line, has been engineered such that the CYP1A1 gene, when activated by an inducer compound, will produce luciferase instead of P450. Eighteen hours after application of an inducer the reaction is stopped by rinsing, the cells lysed, and the cytoplasm measured for luminescence and protein content (for normalization). Induction by such compounds as dioxin, dioxin-like PCB congeners (coplanars), and polycyclic aromatic hydrocarbons (PAHs) infers these xenobiotics are present at levels that are potentially toxic, carcinogenic, or mutagenic to organisms. Multiple wells with attached cells in 2 ml of media were inoculated with various concentrations of toxicants in organic solvents (including DMSO, toluene, and dichloromethane). Volumes tested succes (incomplete abstract) (Anderson et al, 1995)

Study #3

levels of the enzyme Cytochrome P450 1A2 varies 40-fold among individuals and may contribute to cancers caused by heterocyclic amines and other chemicals
rat and human P450 1A2 are 75% identical
some human liver samples are more active (than rat livers stimulated by PCBs) in the activation of carcinogens

Cytochrome P450 1A2 provides an interesting paradigm for inter-individual differences in the metabolism of pro-carcinogens. The enzyme is known to vary 40-fold among individuals and may contribute to cancers caused by heterocyclic amines and other chemicals. Rat and human P450 1A2 are known to be 75% identical and were compared for several catalytic activities. The human enzyme was an order of magnitude more efficient in the N-hydroxylation of two heterocyclic amines. Further, the levels of P450 1A2 expressed in human livers show a 40-fold variation, with some as high as 0.25 nmol P450 1A2 per milligram microsomal protein. Some human liver samples are more active (than those isolated from polychlorinated biphenyl-treated rats) in the activation of heterocyclic amines. A bacterial genotoxicity assay has been developed in which human P450 1A2 and NADPH-P450 reductase are expressed within Escherichia coli and bacterial mutants can be assayed using reversion to lac prototrophy. A random mutagenesis strategy for human P450 1A2 has been developed and used to examine the changes in catalytic activity seen with many single-amino acid substitutions. These results may be of relevance in consideration of genetic polymorphisms. Further, the findings pose a challenge to molecular epidemiology effort in that results with one substrate do not necessarily predict those for others. Some dinitropyrenes are P450 1A2 substrates but others are not. 6-Nitrochrysene can be activated by human P450 1A2 but the (mono) nitropyrenes examined were not; these were oxidized by P450 3A4 instead. (Guerngerich et al, 1999)

Study #4

PCBs enhanced the sensitivity of human liver cancer cells to other toxicants
sensitivity is probably due to increased metabolic conversion of the toxicant to active metabolites after PCB exposure
PCBs induced ECOD enzyme production in the cells, which declined when the PCBs were absent
PCB treated liver cancer cells could detect cytotoxic potencies of 7 different toxins

The neutral red in vitro cytotoxicity assay was adapted for use with the human hepatocellular tumor cell line HepG2 to detect the cytotoxic potencies of polynuclear aromatic hydrocarbons (PAHs). Using benzo[a]pyrene (B[a]P) as the representative PAH, it was determined that a 3-day exposure was the most suitable for detecting cytotoxic potency and that preexposure to 5 micrograms/ml Arochlor enhanced the sensitivity of the HepG2 cells to the toxicant. Such enhanced sensitivity probably reflected increased metabolic conversion of the B[a]P to active metabolites after culturing the cells in the presence of Arochlor. This was shown by a 3-fold increase in the activity of 7-ethoxycoumarin deethylase, an indicator of mixed-function oxygenase activity. Furthermore, a reduction in sensitivity to B[a]P occurred when the cells were cultured in the presence of alpha-napthoflavone, an inhibitor of aryl hydrocarbon hydroxylase activity. When Arochlor-induced cells were transferred to medium lacking Arochlor, the level of 7-ethoxycoumarin deethylase quickly declined to basal levels.Arochlor-induced cells were also able to detect the cytotoxic potencies of benzo[k]fluoranthene, benzo[b]-fluoranthene, chrysene, benzo[a]anthracene pyrene, phenanthrene, and fluoranthene, whereas fluorene, anthracene, acenaphthene, and acenaphthylene were not cytotoxic. (Babich et al, 1988)

Study #5

a single PCB dose after NDMA treatment doubled the number of lung tumors
a single PCB dose after NDMA treatment decreased the number, but increased the size of liver tumors and foci
PCBs also caused complex, multiple effects on NDMA-caused liver tumors (adenomas and carcinomas) and on focal hepatocellular proliferative lesions
bodies retained only 0.1 to 6 ppm PCBs, with 80% being the higher-chlorinated PCBs 153 and 156
NDMA is an industrial and sewage treatment plant by-product, also from auto exhaust and the manufacture of pesticides, rubber tires, alkylamines and dyes.

Polychlorinated biphenyls (PCBs) are widespread, chemically-stable environmental contaminants; some congeners are commonly found in human adipose tissue and breast milk. We investigated the effects of a single dose of one PCB mixture (Aroclor 1254) on tumors initiated by N-nitrosodimethylamine (NDMA), also a common environmental agent. Infant outbred Swiss male mice were treated with NDMA (5 mg/kg) i.p. on the 4th day of life, to initiate lung and liver tumors. Four days later each received a single intragastric dose of PCBs (50, 250, or 500 mg/kg of Aroclor 1254) or oil. Groups were killed 16 and 28 weeks later. At both endpoints the mice given 500 mg/kg PCBs after NDMA developed twice as many lung tumors (alveologenic adenomas) as those treated with NDMA only, a significant difference. The PCBs alone did not cause lung tumors. This is the first demonstration of tumor promotion by PCBs in an extrahepatic organ, and it occurred after a single exposure. There were also complex, multiple effects on NDMA-caused liver tumors (adenomas and carcinomas) and on focal hepatocellular proliferative lesions: PCB treatment after the NDMA was associated with decreased number but increased size of these tumors and foci. All of these changes were accompanied by retention in the bodies of 0.1-6 ppm PCBs, as indicated by gas chromatography with electron capture detection. Of this, 80% or more consisted of 2,4,5,2',4',5'-and 2,3,4,2',4',5'-hexachlorobiphenyls in about equal amounts for periods up to 28 weeks. These results point to a need for both experimental and epidemiological studies of the effect of PCB body burden on tumor development. (Anderson et al, 1986)

Study #6

PCBs are believed to produce liver tumors by promoting clonal outgrowth of initiated cells.
PCBs interact wth specific cellular receptors
receptor binding appears crucial for PCBs’ tumor-causing activity
enzyme-altered foci in rat liver may serve as a sensitive means to estimate the promoting activity of PCBs in rodents

The liver is a major target organ in rodent carcinogenicity assays. Amongst the agents that are effective in producing rodent liver tumours are many chemicals which are not mutagenic, but are believed to mediate their effects by promoting the clonal outgrowth of initiated cells. Some of these chemicals, such as dibenzo-p-dioxins and certain PCBs, have been demonstrated to interact with specific cellular receptors and receptor binding appears crucial for their tumourigenic activity.Enzyme-altered foci in rat liver may serve as a sensitive means to estimate the promoting activity of these agents in rodents. Mechanistic considerations are of relevance when extrapolating these data from rodents to humans. (Schwarz et al, 1995)

Study #7

certain PCBs (#77, 126,169 and 81) induce microsomal aryl hydrocarbon hydroxylase (AHH), bind to the Ah receptor, and enhance microsomal enzyme activities --- similar to the properties of dioxin (TCDD)
the structure of each PCB type determines its toxic effects

The biologic and toxic effects of polychlorinated biphenyls (PCBs) are remarkably dependent on their structure. The most toxic PCBs, namely 3,3',4,4'-tetra-, 3,3',4,4',5-penta- and 3,3',4,4',5,5'-hexachlorobiphenyl are substituted in at least one meta and para position on both phenyl rings (i.e., the lateral positions) and contain no ortho-chloro substituents. These three congeners and a fourth PCB, namely 3,4,4',5-tetrachlorobiphenyl, are approximate isostereomers of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and, in common with TCDD, induce hepatic microsomal benzo[a]pyrene or aryl hydrocarbon hydroxylase (AHH) in rats and rat hepatoma cells in culture. The mono-ortho substituted analogs of the four laterally substituted PCBs also induce microsomal AHH activity and simultaneously enhance microsomal enzyme activities which are inducible by phenobarbitone (PB). This group of PCBs exhibits many of the properties of 2,3,7,8-TCDD and related polychlorinated dibenzo-p-dioxins; there is a close parallel in the relative potencies of these PCBs for AHH induction and their binding affinities for the Ah receptor protein and some of these PCBs are also toxic. Preliminary studies on other halogenated biphenyls confirm that the polarizability of a lateral substituent is an important factor in their activity as AHH inducers (i.e., I greater than Br greater than Cl greater than F). However, preliminary results with other substituted halogenated biphenyls suggest that additional structural factors are also important in determining the activity of these compounds. (Safe et al, 1982)

Study #8

PCBs cause toxic and biologic effects by inducing liver enzymes which activate the metabolism of other chemicals
certain PCBs are more potent inducers and more likely to bind to key cell sites

The relationship between structure and function of polychlorinated biphenyls (PCBs) is discussed. Mechanisms of action are proposed. PCBs are used widely in the manufacture of plasticizers, heat transfer fluids, and flame retardants. Residues are found in water, air, fish, human adipose tissue, blood, and breast milk. PCBs and other halogenated aromatics, such as polychlorinated dibenzo-p-dioxins, polychlorinated dibenzofurans, and polybrominated biphenyls elicit a number of toxic and biological effects as a result of hepatic drug metabolizing enzyme induction. Studies suggest that PCBs substituted in the para and meta positions are the most active inducers of aryl-hydrocarbon-hydroxylase (AHH) and cytochrome-P-450. Quantitative structure/activity relationships are determined by comparing AHH induction potencies of PCBs in rat hepatoma cells and their binding affinities for 2,3,7,8-tetrachlorodibenzo-p-dioxin receptor protein. Results suggest that there is a correlation between induction potency and binding activities. The toxicity of mono ortho chloro and di ortho chloro substituents correlates with biological potency. Effects of coplanar PCBs in responsive C57BL/6J-mice and non responsive DBA/2J-mice are discussed. The difference in response of these inbred strains is attributed to the presence of an Ah receptor in C57BL/6J-mice. The ligand receptor interaction model and correlation with its PCB toxicity is examined. (Safe et al, 1985)

Study #9

liver enzyme stimulation by PCBs played an important role in DNA binding and uptake of carcinogens by cells
binding does not occur if the metabolism is not activated

The compound 2-amino-3-methyl-imidazo(4,5-f)quinoline (IQ) is a powerful mutagenic compound that can induce tumours in different rat and murine target organs (liver, forestomach and small and large bowel). The ability of mutagenic IQ to form adducts to human embryonic enterocyte DNA (intestine 407 cell lines ATCC) has been studied, considering the DNA extracted from the cells and DNA of the in vitro-cultured cells. The activation with the rat hepatic microsomial fraction S9 plays an important role not only in DNA binding, but also in the uptake of IQ by the enterocytes. In the presence of S9 mix, the DNA adduct formation increases with the incubation time; in the absence of metabolic activation, the binding does not occur. Parallel experiments were carried out for comparison on chick embryo hepatocytes; for these cells, the metabolic activation with S9 mix is not as critical as for enterocytes in IQ uptake and DNA binding. [PCBs were used to induce liver microsomal enzyme production.] (Pistolesi et al, 1994)

Study #10

liver and lung tumors started by NDMA were promoted by later treatment with PCBs

Polychlorinated biphenyls (PCB), which are tumor promoters, have been found in human tissues for decades. Their contribution to cancer risk may only now start to appear, due to long human cancer latency and the nature of tumor promotion. Epidemiological associations have been seen between PCB exposure or tissue content and cancer at several sites. In rodents, tumor promotion by PCBs has been little studied in tissues other than liver. Previously, in an experiment modeling infant carcinogen exposure following PCBs received in milk, lung and liver tumors, initiated neonatally in mice by the environmental nitrosamine N-nitrosodimethylamine (NDMA), were promoted by later treatment with Aroclor 1254. The present study was undertaken to confirm and characterize the effects of Aroclor 1254 on tumor number, latency, size and malignancy. Male Swiss mice were given NDMA on postnatal day 4 and Aroclor 1254 (250 mg/kg) on day 8, and killed at intervals. Eight PCB congeners were qua (incomplete abstract) (Anderson et al, 1994)

Study #11

genotoxic activation of 2 tobacco components was catalyzed efficiently by liver microsomes prepared from rats treated with PCBs
genotoxicity of the tobacco extract was observed only after activation by P-450 enzymes

The ability of cigarette smoke condensate to induce a genotoxic response has been measured in liver microsomal and reconstituted monooxygenase systems containing rat and human cytochrome P-450 (P-450) enzymes, as determined by umu gene expression in Salmonella typhimurium TA1535/pSK1002. The reactivities of amino-alpha-carboline and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), two compounds known to be present at considerable levels in cigarette smoke condensate, were also determined and compared with regard to genotoxicity. Amino-alpha-carboline and PhIP are activated principally by P-450 1A2 enzymes in human and rat liver microsomes: (a) activation of both compounds was catalyzed efficiently by liver microsomes prepared from rats treated with 5,6-benzoflavone, isosafrole, or the commercial polychlorinated biphenyl mixture Aroclor 1254, and the activities could be considerably inhibited by antibodies raised against P-450 1A1 or 1A2; (b) the rates of activation of these compounds were correlated with the amount of human P-450 1A2 and of phenacetin O-deethylation activity in different human liver microsomal preparations, and these activities were inhibited by anti-P-450 1A2; (c) reconstituted enzyme systems containing P-450 1A enzymes isolated from rats and humans showed the highest rates of activation of amino-alpha-carboline and PhIP. In rat liver microsomes PhIP may also be activated by P-450 3A enzymes; activity was induced in rats treated with pregnenolone 16 alpha-carbonitrile and was inhibited by anti-human P-450 3A4. However, in humans the contribution of P-450 3A enzymes could be excluded as judged by the very low effects of anti-P-450 3A4 on the microsomal activities and poor correlation with P-450 3A4-catalyzed activities in various liver samples. Cigarette smoke condensate strongly inhibited the activation of several potent procarcinogens by human liver microsomes, particularly the reactions catalyzed by P-450 1A2, but was not so inhibitory of the activation reactions catalyzed by P-450 3A4 and of P-450 2D6-catalyzed bufuralol 1'-hydroxylation. Genotoxic components of the cigarette smoke condensate were extracted by using copper phthalocyanine cellulose (blue cotton). Genotoxicity of this extract was observed only after activation by P-450, and the inhibition of P-450 1A2 activities by these extracts was slight. (Shimada et al, 1991)

Study #12

human liver cancer cells were genetically modified so PCBs would induce a luminescent enzyme, for detection purposes

Samples were collected from Prince William Sound and were extracted by EPA methods (3540, 3550) to produce dichloromethane (DCM) extracts, and small aliquots of these were applied to human liver cancer cells (101L), which produce a luminescent enzyme (luciferase) if dioxins, furans, coplanar PCBs and polycyclic aromatic hydrocarbons (PAHs) are present. Graphs reproduced in black and white. Sponsored by National Marine Fisheries Service, Juneau, AK. Office of Oil Spill Damage Assessment and Restoration., Exxon Valdez Oil Spill Trustee Council, Anchorage, AK. Restoration Office. and National Oceanic and Atmospheric Administration, Auke Bay, AK. Office of Oil Spill. (Anderson et al, 1998)

Study #13

PCB induced enzymes were used to test genotoxicity in N-nitroso-compounds and aromatic amines

Exposure to N-nitroso-compounds and aromatic amines, xenobiotics which require an activation in order to exert their genotoxic potential, is causally associated with gastric cancer. We evaluated the capacity of microsome-containing fractions from human gastric mucosa to activate two model carcinogenic compounds. A 9,000 x g supernatant (S9) was obtained from gastric mucosal specimens and, for comparison, from human liver and aroclor-induced and non induced rat liver. The capacity of the S9 to activate N-nitrosopyrrolidine (NPY) and 2-aminofluorene (2AF) to mutagenic metabolites was tested in the Ames/Salmonella reversion assay, while dimethylnitrosamine (DMN) and aminopyrine (AP) demethylation activities were spectrophotometrically evaluated by using an enzymatic assay of the amount of formaldehyde released following the enzymatic demethylation of the corresponding substrates. Results indicate that human gastric S9 fractions may activate 2AF to a genotoxic derivative and are characterized by DMN and AP demethylase activities higher (p < 0.05) than those of human liver, when expressed in mg/protein (p < 0.05). Although the parameters evaluated can only be considered as a partial measure of the general activating capacity toward dietary and environmental procarcinogens, these results suggest that human gastric mucosa may be directly involved in the metabolic activation of these compounds to mutagenic/carcinogenic species. (Farinati et al, 1991)

Study #14

PCB induced enzymes were used for cancer tests of metabolic extracts of fungi

Twenty strains of the common fungi were isolated from the staple grains in the high incidence liver cancer areas--Fusui county, and their metabolic extracts were prepared. Possible co-mutagenic effects of these metabolic extracts on Salmonella typhimurium TA98 and TA100 mutants were tested. The results showed that in vitro these metabolic extracts had different degrees of co-mutagenic effects. It is considered that these co-mutagenic effects may play an important role in the incidence of liver cancer in Fusui county. [PCBs were used to induce liver microsomal enzyme production.] (Ruan, 1991)

Study #15

PCB induced enzymes were used for cancer tests of benzo(a)pyrene (a fossil-fuel combustion byproduct)

The DNA adducts formed in Salmonella typhimurium when bacteria are incubated with radioactive benzo(a)pyrene and liver microsomal enzymes from several sources has been investigated. When enzyme preparations from Aroclor 1254 or 3-methylcholanthrene induced C57BL/6N (B6) mice were used to mediate activation, the predominant product was n adduct between the 10 position of 7B,8a-dihydroxy-9a,10a epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene and the N-2 position of deoxyguanosine. Similar results were obtained with human liver and with Aroclor-induced rat-liver enzyme preparations. [PCBs were used to induce liver microsomal enzyme production.] (Santella et al, 1979)

Study #16

PCB induced enzymes were used for cancer tests of heterocyclic aromatic amines (HAAs), formed during the cooking of foods

Heterocyclic aromatic amines (HAAs), formed during the cooking of foods, are known to induce tumours in rodent bioassays and may thus contribute to human cancer risk. We tested six HAAs in a morphological transformation assay and in three in vitro genotoxicity assays. The morphological transforming abilities of HAAs were tested, in the presence of rat-liver S9, in the C3H/M2 fibroblast cell line. Concentration levels of 50 microM 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (8-MeIQx), 100 microM 2-amino-3,4,8-trimethylimidazo-[4,5-f]quinoxaline (4,8-DiMeIQx), 50 microM 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 100 microM 2-amino-9H-pyrido[2,3-b]indole (AalphaC), 100 microM 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeAalphaC) and 15 microM 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) induced maximum transformation potencies of 5.5, 6.6, 6.3, 5.2, 7.3 and 9.2 transformed foci per 10(4) surviving cells, respectively. Bacterial mutagenic activity was determined in the presence of rat-liver S9 using the Salmonella typhimurium reverse-mutation assay employing strain YG1019. Mutagenic potencies of 3800 revertants (revs)/ng with 8-MeIQx, 2900 revs/ng with 4,8-DiMeIQx, 3480 revs/ng with IQ, 1.6 revs/ng with AalphaC, 2.9 revs/ng with MeAalphaC and 5 revs/ng with PhIP were observed. Clastogenic activity in vitro was analysed by the micronucleus assay in metabolically competent MCL-5 cells. Dose-dependent induction of micronuclei was observed for all HAAs tested with 1-5.4% of cells containing micronuclei at 10 ng/ml. Micronucleus induction was in the order 4,8-DiMeIQx > 8-MeIQx > IQ > MeAalphaC > PhIP > AalphaC. DNA strand-breaking activity in MCL-5 cells was measured by the alkaline single cell-gel (comet) assay. The lowest effect doses for significant increases (P < or = 0.0007, Mann-Whitney test) in comet tail length (microm) were 45.5 microg/ml (200 microM) for PhIP, 90.9 microg/ml (410-510 microM) for 4,8-DiMeIQx, IQ, MeAalphaC and AalphaC, and 454.5 microg/ml (2130 microM) for 8-MeIQx. It is not yet clear which of these assays most accurately reflects the genotoxic potential to humans of compounds of this class of environmental carcinogens. [PCBs were used to induce liver microsomal enzyme production.] (Pfau et al, 1999)

Study #17

PCB induced enzymes were used for carcinogenicity tests of several foods

We have previously suggested that differences in cancer incidence between Polynesians (including Maoris and people from several Pacific islands) and Europeans in New Zealand may at least partially relate to differences in the species of food plants (fruits, vegetables and cereals) preferentially eaten by these groups. Twenty-five food plants that are typically eaten in different amounts by these two population groups were selected for detailed study. Antimutagenic properties of three extracts from each of the selected plants were investigated using a preincubation mutagenicity assay with Salmonella typhimurium strain TA1538 against the mutagenicity of the heterocyclic amine 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). The data revealed strong antimutagenic properties in several of the food plants commonly eaten by Polynesians, especially rice, watercress, pawpaw, taro leaves, green banana and mango. Using the New Zealand food database, a number of nutrients and micronutrients with antimutagenic and anticarcinogenic potential were identified from the selected food plants. Some of these were tested for antimutagenic potential in parallel experiments to those done with the food plant extracts. Although some of these micronutrients are antimutagens against IQ, their concentrations in the food plants failed to explain the protection against mutagenicity found in the experiments with extracts of the food plants. Thus, other types of chemical, not identified in the database, must be leading to antimutagenesis. Possible active molecules include chlorophylls, carotenoids, flavonoids and coumarins, many of which are also known to be anticarcinogens. If human cancer data are to be interpreted in terms of cancer protection, these components need urgently to be quantified in food plants in the New Zealand diet, especially in those food plants eaten in large amounts by Polynesians. [PCBs were used to induce liver microsomal enzyme production.] (Botting et al, 1999)

Study #18

PCB induced enzymes were used for carcinogenicity tests of chemicals in cooked meats

Heterocyclic aromatic amines are sometimes formed during the cooking of muscle meats, and their mutagenic and carcinogenic effects are of potential concern in the aetiology of human cancer. In a large survey of the heterocyclic amine content of foods, fried or charbroiled hamburgers, fried chicken, chicken breast sandwiches, fish sandwiches and breakfast sausages were purchased from fast-food restaurants. At least three different chains were visited per product and samples from five stores from each chain were pooled. The solid-phase extraction and HPLC method was used to analyse pooled samples for heterocyclic amine content and mutagenic activity with the Ames/Salmonella assay. Samples were analysed in a blind study which also contained quality control samples of two types, one high and one low in heterocyclic amine content and mutagenic activity. Results from the fast-food products showed undetectable levels of heterocyclic amines in 10 of 17 samples and only low levels [ < or = 1 ng/g total of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (DiMeIQx)] in the remaining samples. Compared with literature values based primarily on laboratory and home cooking conditions, fast-food meat products appear to contribute only a small percentage of the estimated daily dietary intake of heterocyclic amines. [PCBs were used to induce liver microsomal enzyme production.] (Knize et al, 1995)

Study #19

PCB induced enzymes were used for anti-mutagenicity tests of several food supplements

Cyclophosphamide (CP), bleomycin (BL), doxorubicin (DOX) and cisplatin (CISP) are potent antitumor drugs used worldwide against many forms of human cancer. As with most such agents, there can be physiological side-effects and the possible induction of mutations and other genotoxic effects in non-tumor cells. It is common for patients to ingest a host of food supplements to diminish the discomforting side-effects of therapy. Because these food supplements are often also rich in antimutagens that could also affect the biological efficacy of the antitumor drugs, we investigated if such antimutagenic agents were indeed antimutagenic to these antitumor drugs. Using the Salmonella/microsome bioassay, we tested CP, BL, DOX, and CP for mutagenicity in the presence and absence of the antimutagens ascorbic acid (AA), chlorophyllin (CHL) and (+)-catechin (CAT). AA was a very effective antimutagen against CISP and less effective against BL and DOX. It was not antimutagenic to CP. CHL was effective as an antimutagen against all four antitumor drugs, and CAT was a strong inhibitor of DOX mutagenicity, but had little effect on BL, CP and CISP. These data now provide a basis for future in vivo antitumor/antimutagen combination studies to determine if these antimutagens function in a manner to reduce genetic effects without having concomitant effects on intended antitumorogenicity of these therapeutic agents. [PCBs were used to induce liver microsomal enzyme production.] (Gentile et al, 1998)

Study #20

liver tumors were significantly increased in number after a single dose of PCBs on day 56 after birth of rats exposed to nitrosamines (in tobacco smoke) in the womb

Both cigarette smoke and nitrosamines have been suggested as contributors to human risk of perinatal cancer initiation. The transplacental (TP) carcinogenicity of NNK was tested in A/J, (C3H/He x C57BL/6)F1, and Swiss (NIH S) mice. Three doses of 100 mg/kg were given ip during the last week of gestation. In A/J offspring killed at 24 wks, 12/66 (18%) had a lung tumor, vs 4/87 (5%) of controls (p less than 0.05). In C3B6F1 male offspring at 72 weeks, TP NNK resulted in 12/30 (40%) with liver tumor vs 8/46 controls (17%) (p less than 0.05). Postnatal treatment of these mice with Na barbital (0.5% in the drinking water) or a single 500 mg/kg dose of a mixture of polychlorinated biphenyls (PCB) did not alter liver tumor numbers. NNK-exposed Swiss male offspring also developed liver tumors, in significantly greater numbers after PCBs given on day 56 (5/26, 19%) vs 3/57 (5%) for NNK only (P = 0.056). Total liver tumor incidence for both NNK groups, 8/83, was significantly greater than for controls, 0/66. The NNK mothers all developed lung tumors in high incidence; the C3H mothers presented liver tumors also. Thus NNK is a weak transplacental carcinogen for sensitive fetal target organs in the mouse. (Anderson et al, 1989)

Study #21

PCB induced enzymes were used to activate the mutagenicity of fluoranthene

Fluoranthene (FA) was studied with respect to possible mechanisms of its high mutagenicity but low carcinogenicity, in comparison with the corresponding properties of benzo[a]pyrene (BaP), and with regard to the synergism of these two compounds shown by van Duuren and Goldschmidt (J Natl Cancer Inst 56, 1976, 1237). FA and BaP activated by S9 fromAroclor 1254 (PCB)-treated rats induce HPRT mutations in CHO cells with about equal effectiveness at the same exposure doses, which also lead to the same frequencies of repairable DNA adducts, enzyme-induced strand breaks being used as an indirect measure of adducts to DNA. FA was also shown to be an efficient inducer of SCE in human peripheral lymphocytes cocultivated with PCB-treated HepG2 cells or with liver cells from PCB-pretreated rats. For the induction of SCE, FA and BaP were shown to act additively. From metabolic studies with liver microsomes from C57Bl/6 mice it is concluded that, whereas BaP induces the metabolism of BaP to the mutagenic epoxide, neither BaP nor FA is able to induce the metabolism of FA. In mutation experiments with V79 cells (XEM2) constitutive for P450 IA1 activity, BaP 7,8-diol but not FA 2,3-diol provokes a high frequency of HPRT mutations. In cells constitutive for P450 IA2 enzymatic activity FA and BaP are but weakly mutagenic and practically nonmutagenic, respectively. Due to the additivity of the genotoxic effects of FA and BaP, induction of an error-prone condition by the latter compound seems to be excluded.(incomplete abstract) (Vaca et al, 1992)

Study #22

PCB induced enzymes were used for cancer and mutation tests of brominated trihalomethanes (found in chlorinated drinking water)

The brominated trihalomethanes (THMs) are mutagenic and carcinogenic disinfection by-products frequently found in chlorinated drinking water. They can be activated to mutagens by the product of the glutathione S-transferase-Theta (GSTT1++-1) gene in Salmonella RSJ100, which has been transfected with this gene. To evaluate this phenomenon in humans, we have examined the genotoxicity of a brominated THM, bromoform (BF), using the Comet assay in human whole blood cultures exposed in vitro. No differences were found in the comet tail length between cultures from GSTT1-1(+) versus GSTT1-1(-) individuals (1.67 +/- 0.40 and 0.74 +/- 0.54 microm/mM, respectively, P = 0.28). The high variability was due to the relatively weak induction of comets by BF. Combining the data from both genotypic groups, the genotoxic potency of BF was 1.20 +/- 0.34 microm/mM (P = 0.003). GSTT1-1 is expressed in red blood cells but not in the target cells (lymphocytes), and expression within the target cell (as in Salmonella RSJ100) may be necessary for enhanced mutagenesis in GSTT1-1(+) relative to GSTT1-1(-) cultures. To examine this, we exposed Salmonella RSJ100 and a control strain not expressing the gene (TPT100) to the most mutagenic brominated THM detected in Salmonella, dibromochloromethane (DBCM), either in the presence or absence of S9 or red blood cells from GSTT1-1(+) or GSTT1-1(-) individuals. S9 did not activate DBCM in the non-expressing strain TPT100, and it did not affect the ability of the expressing strain RSJ100 to activate DBCM. As with S9, red cells from either genotypic group were unable to activate DBCM in TPT100. However, red cells (whole or lysed) from both genotypic groups completely repressed the ability of the expressing strain RSJ100 to activate DBCM to a mutagen. Such results suggest a model in which exposure to brominated THMs may pose an excess genotoxic risk in GSTT1-1(+) individuals to those organs and tissues that both express this gene and come into direct contact with the brominated THM, such as the colon. In contrast, those organs to which brominated THMs would be transported via the blood might be protected by erythrocytes. Such a proposal is reasonably consistent with the organ specificity of drinking water-associated cancer in humans, which shows slightly elevated risks for cancer of the colon and bladder but not of the liver. [PCBs were used to induce liver microsomal enzyme production.] (Landi et al, 1999)

Study #23

PCB induced enzymes were used to test resistance of liver cells to aflatoxin (a natural toxin found in moldy grains)
PCBs did not improve resistance

To evaluate the role of glutathione S-transferase (GST) isoenzymes in induced resistance of hepatocytes to aflatoxin B1 (AFB1), we compared DNA protective activities of different hepatic cytosol preparations and purified GSTs from normal rats, rats exposed to different polychlorinated biphenyls (PCBs), and rats with carcinogen-induced hepatocellular neoplasms, with cytosols or purified GSTs from mouse, rainbow trout, and human livers. These comparisons were performed in an in vitro assay for [3H]AFB1-DNA binding after activation by rat liver microsomes. Cytosol and S-hexylglutathione-affinity-purified GST preparations from livers of mice consistently had strong protective activity against AFB1-DNA binding. The majority of this activity was dependent on the presence of reduced glutathione (GSH) but some GSH-independent protection was observed in mouse hepatic cytosol, but not in purified GST preparations. We found that all of the GSH-dependent DNA-protective activity in mouse liver eluted as a single GST isoenzyme by hydroxyapatite chromatography. Preparations of cytosol and purified GSTs from normal rat liver, rainbow trout liver, and human liver had much less AFB1-specific DNA protective activity than GSTs found in mouse liver preparations. Cytosol from rats with carcinogen-generated liver neoplasms and livers induced with 3,3',4,4'-tetrachlorobiphenyl and 2,2',4,4',5,5'-hexachlorobiphenyl had more GST activity toward CDNB than cytosol from normal rat liver. When equivalent units of GST activity (CDNB) were compared, there was little difference observed between the DNA-protective activities of PCB-induced and normal rat liver cytosols, yet cytosol from rat liver neoplasms was more protective. Purified GST-P (7-7), the GST isoenzyme most induced in carcinogen-generated rat liver neoplasms, was not protective when added at protein concentrations found to be protective for total GSTs isolated from these neoplasms. These studies demonstrate that the resistance of mouse liver to AFB1 can be explained primarily by a single constitutive GST isoenzyme (YaYa or 4-4) with a relatively high activity toward DNA-binding metabolites of AFB1. GST isoenzymes with such high specific DNA protective activity against AFB1 metabolites were not evident in human, rat, or rainbow trout liver or in PCB-induced or neoplastic rat liver preparations. [PCBs were used to induce liver microsomal enzyme production.] (Quinn et al, 1990)

Study #24

acute (large) PCB doses, especially of highly chlorinated PCBs, can increase toxicity of drugs through induction of liver microsomal enzyme P-450.
chronic exposures to low concentrations of PCBs could produce similar responses to drugs
the degree of liver damage correlated with increasing chlorine content of the PCBs

The potentiation of fluroxene (406906) toxicity by polychlorinated-biphenyls (PCB) was studied in rats. Aroclor-1254, Aroclor-1016, Aroclor-1221, or Aroclor-1260 was administered intraperitoneally on 3 days in doses of 100 milligrams per kilogram. Some animals were killed 2 or 4 days after the last injection. Other animals received intraperitoneal injections of undiluted fluroxene in doses of 2.5 grams per kilogram, and were killed 90 minutes or 48 hours later. The rat livers were removed and analyzed for microsomal cytochrome P-450, protein, and the rate of warfarin metabolism. Histological examination also was performed. The more highly chlorinated PCBs induced more of the cytochrome P-450 than the PCBs with fewer chlorine substituents. The destruction of cytochrome P-450 by fluroxene after 90 minutes was markedly enhanced by prior administration of the Aroclors. The proportion of test animals which died after administration of fluroxene increased sharply with the increasing chlorine content of the aroclor given. When fluroxene and PCBs were given together, the degree of liver damage correlated with increasing chlorine content of the PCBs. Aroclor-1016 and Aroclor-1221 produced increases in the rates of 6-hydroxywarfarin and 8-hydroxywarfarin formation from R-warfarin catalyzed Aroclor-1254 induced microsomes. Aroclor-1260 caused a more rapid production of 7-hydroxywarfarin from R-warfarin. The authors conclude that acute doses of the Aroclors, especially those containing large proportions of highly chlorinated-biphenyls, can potentiate the toxicity of administered drugs through induction of the liver microsome P-450. They suggest that chronic exposures to low concentrations of PCBs could produce similar responses to drugs administered to human beings. (Murphy et al, 1979)

Study #25

PCBs ingested with food may play a role in inducing primary cancer of the liver
subliminal quantities of PCBs may play a synergistic (cancer-causing) role with high fat diets

Among the many substances ingested with food which may play a role in inducing primary cancer of the liver are PCBs and organochlorine pesticides (including DDT, aldrin, dieldrin, heptachlor and chlordane). DDT repeatedly administered po was found to produce liver cancer in mice and rats after 2.5 yr. DDTs true carcinogenic effect on humans may be underrated since the effects of its long persistence in the environment remains to be assessed. Residues of the herbicides amitrole and di-allate present in treated crops were found to be hepatocarcinogenic for mice and rats following po and sc administration. Subliminal quantities of potential hepatocarcinogens may play a synergistic role with high fat diets; simultaneous smoking and alcohol intake may have a cocarcinogenic role in the genesis of intestinal, prostate, orophageal and esophageal cancers. (Darnis et al, 1980)

Study #26

PCBs and dioxin potently induced CYP 1A1 enzymes in human liver cancer cells
study used PCB 126

Polychlorinated biphenyls (PCBs) are a group of persistent and widely dispersed environmental pollutants, some of which may be immunotoxic. In the present study, we investigated the effect of PCBs on immune system by assessing apoptotic cell death in human monocytic U937 cells. Among the various congeners tested, 2,2',4,6, 6'-pentachlorobiphenyl (PeCB), a highly ortho-substituted congener, specifically induced DNA fragmentation, a hallmark of apoptosis, while the other examined di-, tri-, tetra-, and pentachlorobiphenyls did not. To further study the 2,2',4,6,6'-PeCB-induced cell death, various features of apoptosis were examined. 2,2',4,6,6'-PeCB caused a decrease in cell viability and induced cellular morphologic features characteristic of apoptosis such as chromatin aggregation and apoptotic bodies. In addition, caspase-3, an executioner of apoptosis, was activated and its substrate, poly(ADP-ribose) polymerase (PARP), was cleaved during 2,2',4,6,6'-PeCB-induced apoptosis. In contrast, 3,3',4,4',5-PeCB, a congener of coplanar structure, as well as 2,3,7,8-TCDD did not induce apoptosis in these human monocytic cells, although they potently induced CYP 1A1 in human hepatoma Hep G2 cells. Taken together, the data indicate that 2,2',4,6,6'-PeCB induces apoptosis in human monocytic cells through a mechanism that is independent of the arylhydrocarbon receptor. This suggests a possibly separate mechanism by which PCBs cause immunosuppression. (Shin et al, 2000)

Study #27

PCBs are known to exert marked effects on the liver
PCBs cause liver cancers in rats and birds, and are suspected of causing cancer in humans
PCB 77 induced DNA adducts in 2 animal and 1 human liver cell lines
Aroclor 1254 failed to produce significant levels of DNA adducts, suggesting that pre-treated cells are required to magnify Aroclor 1254 metabolism

Polychlorinated biphenyls (PCBs) are industrial chemicals which have been detected in fish, birds and humans. They are known to exert marked effects on the liver. They induce hepatocellular carcinoma in rats and birds, and are suspected of being carcinogenic to humans. To better understand the genotoxic effects of PCBs, we used 32P-postlabelling to investigate DNA adduct formation, after exposure to PCBs (Aroclor 1254 and 3,3',4,4'-tetrachlorobiphenyl), in primary cultures of fetal hepatocytes from two animal species and in a human hepatoma cell line (Hep G2). We also studied the induction of 7-ethoxyresorufin-O-deethylase (EROD) in these PCB-treated cells. The three cell types used are known to express different cytochrome P450 families. The aim was to see whether a correlation could be established between EROD activity (a CYP1A1-related activity) and DNA adduct formation. DNA adducts were found in all three models after exposure to 50 microM 3,3',4,4'-tetrachlorobiphenyl. The number of adducts was higher in quail hepatocytes (37 adducts per 10(9) nucleotides) than in rat hepatocytes or Hep G2 cells (20 adducts per 10(9) nucleotides in both cases). The major adduct was the same in all three cell types, but some adducts were found in only one or two species. These inter-species differences probably reflect metabolic differences leading to different ultimate carcinogens. Exposure to Aroclor 1254 failed to produce significant levels of DNA adducts, suggesting that pre-treated cells are required to magnify Aroclor 1254 metabolism. No correlation was found between adduct formation and the level of EROD induction. (Dubois et al, 1995)

Study #28

iron increases PCB induction of liver cancer

The induction of hepatocarcinogenesis by polychlorinated biphenyls (PCBs) in C57BL/10ScSn mice is markedly potentiated by iron. To investigate the effects of iron and PCBs on nuclear populations, C57BL/10ScSn mice received a single dose of iron-dextran (600 mg Fe/kg) and were fed a diet containing 0.01% of the PCBs mixture Aroclor 1254 for up to 6 months. DNA content of isolated nuclei and hepatocytes was estimated by flow cytometry. Cell suspensions and nuclei isolated from Aroclor treated mice after 6 months contained increased diploid (2N) populations compared to controls. In contrast, iron treatment of mice markedly enhanced fractions of octoploid (8N) nuclei by 2 weeks and this effect persisted over the 6 month period. When Aroclor 1254 and iron were administered together there was a synergistic increase in the mononucleated diploid fraction which was significant at 2 weeks and highly significant at 6 months. This became the predominant nuclear effect. At six months, Aroclor 1254 and iron, both alone and in combination, also increased the rate of DNA synthesis in hepatocytes as measured by bromodeoxyuridine (BrdU) incorporation. The chronic polyploidizing effect of iron overload alone was investigated further and shown to be proportional to the dose and was detectable as early as 2 days after 600 mg Fe/kg and 1 week after 150 mg Fe/kg. Polyploidization of nuclei was inhibited by the oral iron chelator CP94. Iron also induced a prolonged reduction in the incidence of binucleated cells. Histologically, nuclear enlargement due to iron was confined to the midzonal region of the liver lobule, whereas iron deposition was greatest in the periportal region. Iron (600 mg/kg) also caused increased nuclear polyploid states in hepatocytes of adult rats and gerbils. Similarly, weanling mice with a dominantly diploid cell population, when treated with iron (300 mg/kg), exhibited a significant shift to a tetraploid (4N) population and a marked increase in proliferation as measured by BrdU incorporation and proliferative cell nuclear antigen (PCNA) detection. These results indicate that Aroclor 1254 and iron induce changes in the mouse hepatocyte population that involve 2N and 8N nuclei respectively. The combination treatment leads to the emergence and proliferation of a mononucleated, diploid population as observed frequently in chemical hepato-carcinogenesis. The reason for the chronic polyploidizing effect of iron is unknown, but may imply both increased DNA synthesis and impairment of nuclear division with implications in human conditions of iron overload.

Study #29

PCBs were known in 1972 to induce liver enzymes and increase liver weight

Recent studies on the toxicity of pesticides and PCBs are reviewed. The findings indicate more or less ubiquitous environmental contamination by low concentrations of organochlorine and diphenyl pesticides. Residues of dieldrin, DDT and its metabolites, heptachlor epoxide and BHC are responsible for most of the contamination by organochlorine pesticides. Recent experiments and studies are concerned primarily with the effects of pesticide residues at the microsomal and mitochondrial levels, on hepatic and endocrine functions, as well as with teratogenic, embryotoxic, and carcinogenic effects. Organochlorine pesticides were found to increase the weight of the liver, and to accelerate the microsomal protein and enzyme synthesis. While there is no irrefutable evidence of the carcinogenicity of organochlorine pesticides in mammals, p,p'-DDT was recently found to increase the incidence of hepatomas in mice when administered in doses of 46.4 mg/kg. The pesticide residue levels are generally too low to induce pathological or even physiological changes in the human organism.

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