|
|
Toxicology. Author manuscript; available in PMC 2008 October 8. Published in final edited form as: Published online 2007 July 7. doi: 10.1016/j.tox.2007.06.102. | PMCID: PMC2063585 NIHMSID: NIHMS30611 |
The Effect of Dietary Glycine on the Hepatic Tumor Promoting Activity of Polychlorinated Biphenyls (PCBs) in Rats Rodica Petruta Bunaciu,1 Job C. Tharappel,1 Hans-Joachim Lehmler,5 Izabela Korwel,5,6 Larry W. Robertson,5 Cidambi Srinivasan,3 Brett T. Spear,1,2,4 and Howard P. Glauert1,2 1Graduate Center for Nutritional Sciences, University of Kentucky, Lexington, Kentucky 40506 2Graduate Center for Toxicology, University of Kentucky, Lexington, Kentucky 40506 3Department of Statistics, University of Kentucky, Lexington, Kentucky 40506 4Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky, Lexington, Kentucky 40506 5Department of Occupational and Environmental Health, University of Iowa, Iowa City, IA 52242-5000 6Department of Environmental Chemistry and Technology, University of Silesia, 40-006 Katowice, Poland Polychlorinated biphenyls (PCBs) are ubiquitious lipophilic environmental pollutants. Some of the PCB congeners and mixtures of congeners have tumor promoting activity in rat liver. The mechanism of their activity is not fully understood and is likely to be multifactorial. The aim of this study was to investigate if the resident liver macrophages, Kupffer cells, are important in the promoting activity of PCBs. The hypothesis of this study was that the inhibition of Kupffer cell activity would inhibit hepatic tumor promotion by PCBs in rats. To test our hypothesis, we studied the effects of Kupffer cell inhibition by dietary glycine (an inhibitor of Kupffer cell secretory activity) in a rat two-stage hepatocarcinogenesis model using 2,2’,4,4’,5,5’-hexachlorobiphenyl (PCB-153, a non-dioxin-like PCB) or 3,3’,4,4’-tetrachlorobiphenyl (PCB-77, a dioxin-like PCB) as promoters. Diethylnitrosamine (DEN, 150 mg/kg) was administered to female Sprague Dawley rats, which were then placed on an unrefined diet containing 5% glycine (or casein as nitrogen control) starting two weeks after DEN administration. On the third day after starting the diets, rats received PCB-77 (300 μmol/kg), PCB-153 (300 μmol/kg), or corn oil by i.p. injection. The rats received a total of 4 PCB injections, administered every 14 days. The rats were euthanized on the 10th day after the last PCB injection, and the formation of altered hepatic foci expressing placental glutathione S-transferase (PGST) and the rate of DNA synthesis in these foci and in the normal liver tissue were determined. Glycine did not significantly affect foci number or volume. PCB-153 did not significantly increase the focal volume, but increased the number of foci per liver, but only in the rats not fed glycine; PCB-77 increased both the foci number and their volume in both glycine-fed and control rats. Glycine did not alter the PCB content of the liver, but did increase the activity of 7-benzyloxyresorufin O-dealkylase (BROD) in liver microsomes from PCB-153 treated rats. However, glycine did not affect the induction of ethoxyresorufin O-dealkylase activity by PCB-77 in liver microsomes. Glycine diminished hepatocyte proliferation in PGST-positive foci, but not in normal tissue. Overall these results do not support the hypothesis that dietary glycine inhibits the promoting activities of PCBs. The observations that PCB-153 increased the number of foci per liver in control rats but not glycine-fed rats and that dietary glycine reduced cell proliferation in PGST-positive foci, however, do not allow us to completely rule out a role for dietary glycine. But the data overall indicate that Kupffer cells likely do not contribute to the tumor promoting activities of PCB-77 and PCB-153. Keywords: Kupffer cells, glycine, 2,2’,4,4’,5,5’-hexachlorobiphenyl, 3,3’,4,4’-tetrachlorobiphenyl, tumor promotion, hepatocyte proliferation Polychlorinated biphenyls (PCBs) are ubiquitous lipophilic environmental pollutants which tend to accumulate into the food chain and to concentrate toward the top of this chain, i.e. into humans. At least some of the PCB congeners and mixtures are hepatic tumor promoters in rats. As reviewed by Glauert et al. (2001), strong evidence shows that PCB mixtures and medium- and highly-chlorinated individual congeners are hepatic tumor promoters in rats. The rodent model is considered a very good indicator of potential hepatic damage in humans ( Hayes et al. 1982) . Seven studies in rats revealed that PCBs promote gross liver tumors and 42 studies demonstrated that PCBs promote altered hepatic foci in rats ( Dean et al. 2002; Glauert et al. 2005; Glauert et al. 2001; Tharappel et al. 2002; Whysner and Wang 2001). Epidemiological studies, however, reached variable conclusions. Some studies showed that overall mortality rates are not higher in PCB exposed groups than in non-exposed groups ( Bosetti et al. 2003; Kuratsune et al. 1987; Sinks et al. 1992). Some studies find no association between cancer mortality and PCBs ( Kimbrough et al. 2003; Rusiecki et al. 2004). But other data suggest a correlation between PCB exposure and carcinogenesis. A study performed in Poland found that adipose tissue samples of patients who have died of liver cancer contained 4.7 μg/g of PCBs (23% of which was 2,2’,4,4’,5,5’-hexachlorobiphenyl [PCB-153]), while in samples from cancer-free subjects, the level was less then 1.9 μg/g of PCBs ( Falandysz et al. 1994) . Twelve deaths among 2061 victims of the Yucheng 1979, Taiwan rice-oil poisoning, 3.5 years after the accident were due to hepatoma, liver cirrhosis, or liver diseases with hepatomegaly ( Hsu et al. 1985). Autopsies of 12 Yusho patients revealed five cases of carcinomas: two of the liver, two of the lung, and one of the esophagus ( Kikuchi 1984). Another study on Yusho patients reported that in males the incidence of liver cancer was significantly higher in those patients than in unexposed males ( Kuratsune et al. 1987). Synergistic factors or different congener composition may account for the different conclusions of the conflicting studies. The mechanisms underling the correlation between PCBs and cancer are not fully understood and might be multifactorial. PCB-153 is one of the most abundant congeners in the environment, and in both animals and humans ( Chu et al. 2003; Duarte-Davidson et al. 1994; Falandysz et al. 1994; Gill et al. 2004; Lanting et al. 1998; Mariottini et al. 2000). Besides being one of the most abundant congeners in the environment, PCB-153 has been previously shown to increase hepatocyte proliferation in rats ( Lu et al. 2003). PCB-77 is present in some dietary supplements for human consumption, such as cod liver oil ( Storelli et al. 2004). In addition, PCB-77 was present in much higher concentration than PCB-126 and PCB-169 in human adipose tissue samples from Concepción, Chile ( Mariottini et al. 2000). Both PCB-77 and PCB-153 have tumor promoting activity ( Glauert et al. 2001). The mechanisms of their promoting activities are still unclear, though one possibility is an imbalance between increased cell replication and suppressed apoptosis in the hepatocyte population. Most of the PCB congeners do not directly interact with DNA, but they may promote or amplify in the hepatocyte the consequences of mutagenic events that would otherwise be repaired. The increased cell proliferation and decreased apoptosis increase the probability that a spontaneous mutation could be propagated and that successive mutations accumulate into the genotype of some cells with a net result of promotion and progression of a tumor. We have hypothesized that the activation of Kupffer cells by PCB-153 and PCB-77 is necessary for the formation of hepatic preneoplastic foci. Our working model is that PCBs activate Kupffer cells. It is known that Kupffer cells, when activated, release factors (such as TNF-α, HGF, IL-1β, and PGE 2), which can cause tissue injury and altered gene expression in hepatocytes, leading to an increased mitogenic rate ( CIimuro et al. 1997; Edwards et al. 1993). Exacerbated Kupffer cell activation might have multiple causes, one being the presence of xenobiotic toxic compounds, as in the case of hepatotoxicity induced by peroxisome proliferators. Peroxisome proliferators are a class of nongenotoxic carcinogens. Wy-14,643, a member of this class, activates NF-κB in the Kupffer cells ( Rose et al. 2000), and stimulates the Kupffer cells to release mitogenic cytokines such as TNF-α ( Rose et al. 1997b) and reactive species such as superoxide ( Rose et al. 1999b; Rusyn et al. 2000). To test our hypothesis of Kupffer cell involvement in tumor promotion, we studied the effects of Kupffer cell inhibition on two-stage rat hepatocarcinogenesis using PCB-153 (a non-coplanar PCB) or PCB-77 (a coplanar PCB) as promoters. Diethylnitrosamine (DEN, 150 mg/kg) was used as the initiator. There are several ways to inhibit Kupffer cell secretory activity, including dietary administration of the amino acid glycine ( Ikejima et al. 1996; Rose et al. 1997a), and we chose this model. Glycine or casein (as nitrogen control) was fed in an unrefined diet at 5% starting two weeks after DEN administration. The rats received four biweekly i.p. injections of vehicle (corn oil) or of PCB-77 or PCB-153. We quantified the formation of preneoplastic focal lesions expressing placental glutathione S- transferase (PGST) and the rate of DNA synthesis in these foci and in the normal tissue. Chemicals PCB-153 (2,2’,4,4’,5,5’-hexachlorobiphenyl) and PCB-77 (3,3’,4,4’-tetrachlorobiphenyl) were synthesized and characterized as described previously ( Lehmler and Robertson 2001; Schramm et al. 1985). The purity of each PCB congener was greater than 99%, as assayed by gas-chromatography. Vitamin E-stripped corn oil was obtained from Acros Organics (Morris Plains, NJ). The diet for the entire study was Harlan Teklad, Global 18% protein Rodent Diet 2918 (Madison, WI). Glycine was from Sigma Chemical Company (St. Louis, MO). Casein was from Teklad (Madison, WI). Alzet osmotic pumps (model 2ML1) were obtained from Alza Scientific Products (Palo Alto, CA). The anti-5-bromo-2’-deoxyuridine (BrdU) antibody was purchased from Becton-Dickinson (San Jose, CA). Anti-placental glutathione S-transferase (PGST) antibody was from Novocastra (New Castle upon Tyne, England). Vectastain ABC Kit was from Vector Laboratories, (Burlingame, CA). The Antigen Retrieval Citra solution was purchased from BioGenex (San Ramon, CA). All other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). Experimental Design Fifty female Sprague-Dawley rats weighing 100-125g at arrival (Harlan Sprague Dawley, Indianapolis, IN), were housed three per hanging-wire, stainless-steel cage in a temperature- and light-controlled room and were fed a powdered irradiated unrefined rodent diet (Harlan Teklad, Global 18% protein Rodent Diet 2918) and water ad libitum. The experimental protocols and procedures that involved rats were approved by the Institutional Animal Care and Use Committee of the University of Kentucky and were in accordance to all policies for the use and care of laboratory research animals as stipulated by the NIH. Upon arrival, the animals were allowed to adjust for 1 week before starting the experiment. DEN was dissolved in saline and administrated by gavage, 150 mg/kg body weight. This dose was chosen based on previous studies ( Berberian et al. 1995; Glauert et al. 2005; Stemm et al. 2005; Tharappel et al. 2002) Glycine or casein (casein contains 1.8% glycine) was fed in the unrefined diet at 5% starting on the 14th day after DEN initiation until the end of the experiment. Glycine was fed at the 5% level because of previous studies showing that this level inhibited the induction of cell proliferation and hepatic tumors by peroxisome proliferators ( Rose et al. 1999a; Rose et al. 1997a). The diet was administered ad libitum in glass feeding cups. On the third day after starting the diets, PCB-77 (300 μmol/kg), PCB-153 (300 μmol/kg), or corn oil were injected i.p.; the number of rats in each group is shown in Table 1. Both PCB-77 and PCB-153 have been shown to have promoting activity in rat liver using a dose of 300 μmol/kg ( Glauert et al. 2001). The rats received a total of 4 PCB injections, administered every 14 days. Euthanasia was by overexposure to carbon dioxide, on the 10th day after the last PCB injection. 72 hours before the euthanasia, the rats were surgically implanted with Alzet osmotic pumps containing BrdU (20mg BrdU/mL PBS, 10 μL/h) for the measurement of DNA synthesis. The livers were immediately removed and weighed; a portion of each was frozen in liquid nitrogen and stored at −80°C until used. For each rat, a piece of liver was randomly taken from 4 different lobes and fixed in 10% buffered formalin. | Table 1Effect of PCBs and glycine on the body weight, liver weight and relative liver weight |
PCB Analysis PCBs 77 and 153 were extracted from liver samples using an Accelerated Solvent Extractor ASE 200 (Dionex Corporation, Sunnyvale, CA) as described previously ( Kania-Korwel et al. 2007). A recovery standard solution consisting of PCBs 14, 65, and 166 (50 μL; 4 mg PCB/L in hexane) was added to the extraction cells prior to extraction using hexane:acetone. The PCB extract was concentrated to 1 mL and an internal standard solution consisting of PCBs 30 and 204 (300 μL; 100.8 μg PCB/L in hexane) was added. Sulfur impurities were removed using standard EPA procedures prior to PCB analysis ( Kania-Korwel et al. 2005; Kania-Korwel et al. 2007; Lehmler et al. 2003). The PCB analysis was performed using a HP 6890 gas chromatograph with 60-m DB-5 capillary column and a 63Ni μ-ECD detector ( Kania-Korwel et al. 2005). The detector temperature was 300°C. The oven temperature program was as follows: 100°C held for 1 min, then increased by 1°/min from 100 to 240°C, 10°/min from 240 to 280°/min, hold for 20 min. An aliquot of the PCB extract (4 μL) was injected and the concentration of PCB 77 and 153 in each sample was determined using the internal standard method. Method blanks (samples containing only Celite and Florisil) and tissue blanks (liver tissue samples from control animals) were analyzed with each set of samples. The PCB 77 and PCB 153 levels in the method blanks were 8±5 and 4±2 ng, respectively. The recoveries rates of the surrogate standards were 98±9.0%, 94±11%, and 90±11% for PCBs 14, 65 and 165, respectively. Corrections of total PCB concentration were made for recoveries < 100% using the recovery rates of PCB 65. The method detection limit for PCB 77 was 16 ng, and for PCB 153 6 ng. The minimum limit of detection was for PCB 77 52 ng, and for PCB 153 20 ng. Lipid Content Determination Lipids were extracted from tissue samples with a chloroform:methanol mixture (2:1 v/v) using an Accelerated Solvent Extractor ASE 200 (Dionex Corporation, Sunnyvale, CA) as described previously ( Kania-Korwel et al. 2007). The lipid content was determined gravimetrically after evaporation of the solvents and used to calculate lipid adjusted PCB levels in the liver. Alkoxyresorufin O-dealkylation Assay The microsomal fraction was isolated from the whole-liver homogenate using the method described by Schramm et al. (1985). Microsomal 7-benzyloxyresorufin and ethoxyresorufin were used as specific substrates for the CYP2B1/2 and CYP1A1 isozymes, respectively, in the alkoxyresorufin O-dealkylase method ( Burke and Mayer 1974).The absorbance of resorufin was detected with a fluorescence spectrophotometer at an excitation wavelength of 556 nm and an emission wavelength of 589 nm. Immunohistochemical Staining Tissues were double-stained for PGST and for BrdU. The formalin-fixed liver tissues were paraffin-embedded, sectioned, laid on glass slides, stained with an anti-PGST antibody to identify altered hepatic foci (using Vector ® Red as the staining agent), stained with an anti-BrdU antibody to identify nuclei that had incorporated BrdU (using diaminobenzidine (DAB) as the staining agent), and counter-stained with hematoxylin ( Wynford-Thomas and Williams 1986)The Vectastain ABC Kit (Vector Laboratories, Burlingame, CA) was used, according to the protocol provided by the manufacturer. Three lobes per each slide were counted randomly, using at least 1000 hepatocellular nuclei per lobe and not less than 3500 total per slide, with an average of 400 nuclei being from PGST-positive foci. The labeling index was expressed as the percentage of number of labeled hepatocyte nuclei out of total number of hepatocyte nuclei and was calculated separately for focal and non-focal hepatocytes. Analysis of PGST-Positive Foci The number and volume of PGST-positive foci were measured using a computer digitizing system developed at University of Wisconsin ( Campbell et al. 1982; Campbell et al. 1986; Xu et al. 1998). The images were captured using a Nikon Eclipse E800 microscope equipped with MACRO 0.5x and 1.0x lenses. The number of foci/cm 3 (Saltykov method), foci/liver (Saltykov method), the mean focal volume (Saltykov method), and the volume fraction (Delesse method) were quantified. Statistical Analysis Most of the data, including those for the body and liver weights, labeling index, mean focal volume, and volume fraction, were analyzed by 2 × 3 analysis of variance (ANOVA). However, data for PCB levels were analyzed by 2 × 2 ANOVA because PCB-77 was not quantified in PCB-153-treated rats and PCB-153 was not quantified in PCB-77-treated rats. Individual differences between means were determined using the Bonferroni post hoc test. The number of foci per liver and number of foci per cubic centimeter data were analyzed by negative binomial regression model with logarithm as the link function. The statistical comparison was made between the PCB treatment groups with the respective corn oil groups (control groups), separately for casein and glycine diets. The goodness of fit of the model was assessed by Pearson X 2 value adjusted for overdispersion and the parameters of the model were estimated by the method of maximum likelihood. The Wald’s asymptotic procedure was used to determine the p values for significance of the differences between the PCB treatment groups and the control groups. The statistical software PROC GENMOD of SAS, version 8, was used to carry out the mentioned statistical analyses. We used the negative binomial regression model for the analysis of data obtained for the number of foci per liver and number of foci per cubic centimeter because of the discrete nature of these measurements ( Espandiari et al. 2003). SAS version 8 (SAS Institute, Cary, NC) was used for the above analysis. The results were expressed as means ± standard deviations (SD). The results were considered significant at p<0.05. The effects of dietary glycine on two-stage hepatocarcinogenesis promoted by PCB-153 and PCB-77 were assessed in female Sprague-Dawley rats initiated with DEN. The body weights were not significantly modified by any of the treatments ( Table 1). The absolute and relative liver weights were significantly increased in the rats treated with PCB-77 (p<0.05) but not with PCB-153, and the glycine treatment had no effect ( Table 1). The livers of control animals (which did not receive PCBs) contained detectable background exposure of about 50 ng/g wet weight of PCB-77 and PCB-153. These values were significantly lower than those of the PCB treated groups ( Table 2). With the same dosage, the PCB-153 treated animals have about 2.5 times higher respective PCB levels than animals treated with PCB-77. However, dietary glycine did not significantly affect hepatic PCB levels. | Table 2Effect of PCBs and glycine on hepatic PCB concentrations |
The activities of the cytochrome P-450 1A and 2B families were examined by quantifying EROD and BROD activities, respectively. BROD and EROD activities were induced by PCB-153 and PCB-77, respectively (). Dietary glycine significantly augmented the PCB-153-induced increase in BROD activity, but had no significant effect on PCB-77-induced EROD activity. PCB-77 significantly increased the number of PGST-positive foci per liver and per cubic centimeter as well as the mean focal volume and the total focal volume as a percentage of liver volume, both in the casein and in the glycine groups ( Table 3.). PCB-153 significantly increased the number of foci per liver compared to the corn oil group for the casein diet only. The number of foci per cubic centimeter was slightly increased for the casein diet by PCB-153 (p= 0.08) but not for the glycine diet. PCB-153 did not significantly increase the mean focal volume or the percentage of liver occupied by foci. There were no significant differences in focal number or volume between the casein and glycine diets in rats administered vehicle, PCB-77, or PCB-153. | Table 3Effect of PCBs and glycine on the induction of PGST-positive foci |
To measure hepatocyte proliferation in normal and PGST-positive hepatocytes, we quantified the labeling indexes after 3 days of continuous BrdU infusion using Alzet pumps. In normal hepatocytes, the BrdU labeling index was similar among all groups (). In PGST-positive hepatocytes, rats fed glycine had a lower labeling index, but neither PCB affected the labeling index. The labeling index was not different between different lobes of the same animal, regardless of the treatment. As expected, cell proliferation was higher in the PGST-positive foci than in the normal tissue. The importance of this study resides in the fact that human exposure to PCBs (especially PCB-153) is ubiquitous and once the PCBs are in the body, important quantities of the parent congeners or their metabolites are retained for long periods of time ( Chu et al. 2003; Guo et al. 1997). Therefore, the real human health concern is the long term effects of PCBs in the body, such as cancer. In this study, we tested the hypothesis that the secretory activity of Kupffer cell is required for the promoting activity of polychlorinated biphenyls. For this, we examined the ability of dietary glycine to inhibit PCB-induced tumor promotion, since several studies have shown that glycine decreases the production of superoxide and TNF-α by Kupffer cells ( Ikejima et al. 1996; Rose et al. 1999a; Rose et al. 1997a; Rusyn et al. 1999). The inhibition by glycine of TNF-α production by Kupffer cells is not as strong as using agents such as gadolinium chloride or methyl palmitate, but that may be an advantage for a therapeutic agent ( Rentsch et al. 2005). Kupffer cell inhibition by glycine has also been shown to affect lipid metabolism in the liver ( Neyrinck 2004). There were no adverse effects of glycine in this study, based on changes in body weight. However, since glycine receptors are present in other tissues, for example the central nervous system ( Cascio 2004; Rajendra et al. 1997), it is likely that the administration of high levels of glycine exerted other biochemical effects. More recently it has been shown that glycine might affect tumor promotion by inhibiting angiogenesis ( Yamashina et al. 2005). However, the ability of glycine to inhibit the production of cytokines such as TNF-α was not measured in this study. Therefore, any changes in glycine-fed rats cannot be unequivocally linked to lower levels of TNF-α or other cytokines, and the lack of an effect of glycine on altered hepatic foci production could be due to a lack of an effect on the levels of TNF-α or other cytokines. Our present data show that the PCB treatment leads to increased PCB congener levels in the liver and increased cytochrome P-450 related enzyme activities, showing that the administration of the PCBs brought about the expected biochemical changes in the liver. With the same dosage, the PCB-153 treated animals have about 2.5 times higher respective PCB levels than animals treated with PCB-77. This difference is to be expected since PCB-77 is metabolized faster than PCB-153 ( Robertson and Hansen 2001) . Glycine increased cytochrome P-450 activity only for PCB-153. It was reported that, if Kupffer cells are co-cultured with hepatocytes and treated with LPS (a well known Kupffer cell activator) and simultaneously treated with phenobarbital, the induction by phenobarbital of cytochrome P-450 2B1 mRNA and the activity of PROD are significantly reduced (85%) ( Milosevic et al. 1999). In our experiment, the activity of cytochrome P-450 2B1/2 was increased by PCB-153 + glycine by about 50% compared to PCB-153 + casein. Cytokines, released by macrophages, are possibly responsible for the down-regulation of cytochrome P-450 2B1/2B2 activities ( Milosevic et al. 1999). Reports show that TNF-α, IL-1β , IL-2 are able to reduce cytochrome P-450 2B1 and 1A1 levels ( Cantoni et al. 1995; Ferrari et al. 1992; Milosevic et al. 1999; Pous et al. 1990). In this study, the numbers of PGST-positive foci were significantly increased by both PCBs but were not significantly affected by dietary glycine. PCB-153 increased the number of foci per liver in the casein-fed group but not in the glycine-fed group. The volume of foci was increased by PCB-77 but was not affected by PCB-153 or dietary glycine. Cell proliferation was not increased by PCB treatment in this study either in the normal hepatocytes or in the PGST-positive hepatocytes. As expected, cell proliferation was higher in the PGST-positive foci than in the normal tissue. Previous studies using the same protocol on rats on the same strain, sex and age ( Berberian et al. 1995; Glauert et al. 2005; Tharappel et al. 2002) found that cell proliferation in both focal and nonfocal hepatocytes was increased by PCB-77 (unlike our study) but was not affected by PCB-153 (similar result to our study). In summary, in this study we have examined the hypothesis that Kupffer cell inactivation by dietary glycine would inhibit the promoting activities of PCB congeners. This study is the first to examine if Kupffer cell activation is important in the promoting activity of PCBs. Our findings indicate that dietary glycine likely does not influence the promoting activities of PCB-77 and PCB-153; however, the observations that PCB-153 increased the number of foci per liver in control rats but not glycine-fed rats and the reduction in cell proliferation in PGST-positive foci in glycine-fed rats do not allow us to completely rule out a role for dietary glycine. But the data overall indicate that Kupffer cells likely do not contribute to the tumor promoting activities of PCB-77 and PCB-153. These results clearly differ from those of Rose et al. (1999a), who found that Kupffer cell inactivation by dietary glycine inhibited liver carcinogenesis by the peroxisome proliferator Wy-14,643. The findings of the present study imply that Kupffer cell activation may not be essential for hepatic tumor promotion. In studies published thus far, PCBs (present study) and peroxisome proliferators ( Rose et al., 1999a) are the only hepatic tumor promoting agents to be examined in Kupffer cell inactivation models. Only the study of other hepatic tumor promoting agents will provide further evidence as to whether Kupffer cell activation is important for most tumor promoting agents, with PCBs being an exception, or whether it is only important in promotion by peroxisome proliferators. We are grateful to Dr. Chantal A. Rivera (Baylor College of Medicine, TX) and Dr. Ion Deaciuc (University of Louisville, KY) for useful information regarding the glycine model. This research was supported by NIEHS (ES07380, ES012475, ES013661) and the Kentucky Agricultural Experiment Station. R.P. Bunaciu was supported by the Training Core of the Superfund Basic Research Program (ES07380). | | BrdU | bromodeoxyuridine | | | BROD | Benzyloxyresorufin O-dealkylase | | | DEN | diethylnitrosamine | | | EROD | Ethyoxyresorufin O-dealkylase | | | LPS | lipopolysaccaride | | | PCBs | polychlorinated biphenyls | | | PCB-153 | 2,2’,4,4’,5,5’-hexachlorobiphenyl | | | PCB-77 | 3,3’,4,4’-tetrachlorobiphenyl | | | PGST | placental glutathione S- transferase |
- Berberian I, Chen L-C, Robinson FR, Glauert HP, Chow CK, Robertson LW. Effect of dietary retinyl palmitate on the promotion of altered hepatic foci by 3,3’,4,4’-tetrachlorobiphenyl and 2,2’,4,4’,5,5’hexachlorobiphenyl in rats initiated with diethylnitrosamine. Carcinogenesis. 1995;16:393–398. [PubMed]
- Bosetti C, Negri E, Fattore E, La Vecchia C. Occupational exposure to polychlorinated biphenyls and cancer risk. Eur J Cancer Prev. 2003;12:251–255. [PubMed]
- Burke MD, Mayer RT. Ethoxyresorufin: direct fluorimetric assay of a microsomal O-dealkylation which is preferentially inducible by 3-methylcholanthrene. Drug Metabolism and Disposition. 1974;2:583–588. [PubMed]
- Campbell HA, Pitot HC, Potter VR, Laishes BA. Application of quantitative stereology to the evaluation of enzyme-altered foci in rat liver. Cancer Res. 1982;42:465–472. [PubMed]
- Campbell HA, Xu YD, Hanigan MH, Pitot HC. Application of quantitative stereology to the evaluation of phenotypically heterogeneous enzyme-altered foci in the rat liver. J Natl Cancer Inst. 1986;76:751–767. [PubMed]
- Cantoni L, Carelli M, Ghezzi P, Delgado R, Faggioni R, Rizzardini M. Mechanisms of interleukin-2-induced depression of hepatic cytochrome P-450 in mice. Eur J Pharmacol. 1995;292:257–263. [PubMed]
- Cascio M. Structure and function of the glycine receptor and related nicotinicoid receptors. J Biol Chem. 2004;279:19383–19386. [PubMed]
- Chu S, Covaci A, Schepens P. Levels and chiral signatures of persistent organochlorine pollutants in human tissues from Belgium. Environmental Research. 2003;93:167–176. [PubMed]
- CIimuro Y, Gallucci RM, Luster MI, Kono H, Thurman RG. Antibodies to tumor necrosis factor alfa attenuate hepatic necrosis and inflammation caused by chronic exposure to ethanol in the rat. Hepatology. 1997;26:1530–1537. [PubMed]
- Dean CE, Jr, Benjamin SA, Chubb LS, Tessari JD, Keefe TJ. Nonadditive hepatic tumor promoting effects by a mixture of two structurally different polychlorinated biphenyls in female rat livers. Toxicol Sci. 2002;66:54–61. [PubMed]
- Duarte-Davidson R, Wilson SC, Jones KC. PCBs and other organochlorines in human tissue samples from the Welsh population: I--adipose. Environmental Pollution. 1994;84:69–77. [PubMed]
- Edwards MJ, Keller BJ, Kauffman FC, Thurman RG. The involvement of Kupffer cells in carbon tetrachloride toxicity. Toxicol Appl Pharmacol. 1993;119:275–279. [PubMed]
- Espandiari P, Glauert HP, Lehmler HJ, Lee EY, Srinivasan C, Robertson LW. Polychlorinated biphenyls as initiators in liver carcinogenesis: resistant hepatocyte model. Toxicol Appl Pharmacol. 2003;186:55–62. [PubMed]
- Falandysz J, Yamashita N, Tanabe S, Tatsukawa R. Congener-specific data of polychlorinated biphenyl residues in human adipose tissue in Poland. The Science of The Total Environment. 1994;149:113–119. [PubMed]
- Ferrari L, Kremers P, Batt AM, Gielen JE, Siest G. Differential effects of human recombinant interleukin-1 beta on cytochrome P-450-dependent activities in cultured fetal rat hepatocytes. Drug Metab Dispos. 1992;20:407–412. [PubMed]
- Gill U, Covaci A, Ryan JJ, Emond A. Determination of persistent organohalogenated pollutants in human hair reference material (BCR 397): an interlaboratory study. Analytical and Bioanalytical Chemistry. 2004;380:924–929. [PubMed]
- Glauert HP, Lu Z, Kumar A, Bunaciu RP, Patel S, Tharappel JC, Stemm DN, Lehmler HJ, Lee EY, Robertson LW, Spear BT. Dietary vitamin E does not inhibit the promotion of liver carcinogenesis by polychlorinated biphenyls in rats. J Nutr. 2005;135:283–286. [PubMed]
- Glauert HP, Robertson LW, Silberhorn EM. PCBs and tumor promotion. In: Robertson LW, Hansen LG, editors. PCBs: Recent Advances in Environmental Toxicology and Health Effects. University Press of Kentucky; Lexington: 2001. pp. 355–371.
- Guo YL, Ryan JJ, Lau BP, Yu ML, Hsu CC. Blood serum levels of PCBs and PCDFs in Yucheng women 14 years after exposure to a toxic rice oil. Arch Environ Contam Toxicol. 1997;33:104–108. [PubMed]
- Hayes AW, Fedorowski T, Balazs T, Carlton WW, Fowler BA, Gilman MR, Heyman I, Jackson BA, Kennedy GL, Shapiro RE, Smith CC, Tardiff RG, Weil CS. Correlation of human hepatotoxicants with hepatic damage in animals. Fundam Appl Toxicol. 1982;2:55–66. [PubMed]
- Hsu S, Ma C, Hsu S, Wu S, Hsu N, Yeh C, Wu S. Discovery and epidemiology of PCB poisoning in Taiwan: a four-year followup. Environ Health Perspect. 1985;59:5–10. [PubMed]
- Ikejima K, Iimuro Y, Forman DT, Thurman RG. A diet containing glycine improves survival in endotoxin shock in the rat. Am J Physiol. 1996;271:G97–103. [PubMed]
- Kania-Korwel I, Hornbuckle KC, Peck A, Ludewig G, Robertson LW, Sulkowski WW, Espandiari P, Gairola CG, Lehmler H-J. Congener specific tissue distribution of Aroclor 1254 and a highly chlorinated environmental PCB mixture in rats. Environ Sci Technol. 2005:3513–3520. [PubMed]
- Kania-Korwel I, Shaikh NS, Hornbuckle KC, Robertson LW, Lehmler H-J. Enantioselective disposition of PCB 136 (2,2’,3,3’,6,6’-hexachlorobiphenyl) in C57BL/6 mice after oral and intraperitoneal administration. Chirality. 2007;19:56–66. [PubMed]
- Kikuchi M. Autopsy of patients with yusho. Prog Clin Biol Res. 1984;137:19–30. [PubMed]
- Kimbrough RD, Doemland ML, Mandel JS. A mortality update of male and female capacitor workers exposed to polychlorinated biphenyls. J Occup Environ Med. 2003;45:271–282. [PubMed]
- Kuratsune M, Ikeda M, Nakamura Y, Hirohata T. A cohort study on mortality of “yusho” patients: a preliminary report. Princess Takamatsu Symp. 1987;18:61–66. [PubMed]
- Lanting CI, Huisman M, Muskiet FAI, Van Der Paauw CG, Essed CE, Boersma ER. Polychlorinated biphenyls in adipose tissue, liver, and brain from nine stillborns of varying gestational ages. Pediatric Research. 1998;44:222–225. [PubMed]
- Lehmler H-J, Price DJ, Garrison AW, Birge WJ, Robertson LW. Distribution of PCB 84 enantiomers in C57Bl/6 mice. Fres Environ Bull. 2003;12:254–260.
- Lehmler HJ, Robertson LW. Synthesis of polychlorinated biphenyls (PCBs) using the Suzuki-coupling. Chemosphere. 2001;45:137–143. [PubMed]
- Lu Z, Tharappel JC, Lee EY, Robertson LW, Spear BT, Glauert HP. Effect of a single dose of polychlorinated biphenyls on hepatic cell proliferation and the DNA binding activity of NF-kappaB and AP-1 in rats. Mol Carcinog. 2003;37:171–180. [PubMed]
- Mariottini M, Aurigi S, Focardi S. Congener profile and toxicity assessment of polychlorinated biphenyls in human adipose tissue of Italians and Chileans. Microchemical Journal. 2000;67:63–71.
- Milosevic N, Schawalder H, Maier P. Kupffer cell-mediated differential down-regulation of cytochrome P450 metabolism in rat hepatocytes. Eur J Pharmacol. 1999;368:75–87. [PubMed]
- Neyrinck A. Modulation of Kupffer cell activity: physio-pathological consequences on hepatic metabolism. Bull Mem Acad R Med Belg. 2004;159:358–366. [PubMed]
- Pous C, Giroud JP, Damais C, Raichvarg D, Chauvelot-Moachon L. Effect of recombinant human interleukin-1 beta and tumor necrosis factor alpha on liver cytochrome P-450 and serum alpha-1-acid glycoprotein concentrations in the rat. Drug Metab Dispos. 1990;18:467–470. [PubMed]
- Rajendra S, Lynch JW, Schofield PR. The glycine receptor. Pharmacol Ther. 1997;73:121–146. [PubMed]
- Rentsch M, Puellmann K, Sirek S, Iesalnieks I, Kienle K, Mueller T, Bolder U, Geissler E, Jauch KW, Beham A. Benefit of Kupffer cell modulation with glycine versus Kupffer cell depletion after liver transplantation in the rat: effects on postischemic reperfusion injury, apoptotic cell death graft regeneration and survival. Transpl Int. 2005;18:1079–1089. [PubMed]
- Robertson LW, Hansen LG. PCBs: Recent Advances in Environmental Toxicology and Health Effects. University Press of Kentucky; Lexington: 2001.
- Rose ML, Cattley RC, Dunn C, Wong V, Li X, Thurman RG. Dietary glycine prevents the development of liver tumors caused by the peroxisome proliferator WY-14,643. Carcinogenesis. 1999a;20:2075–2081. [PubMed]
- Rose ML, Germolec D, Arteel GE, Schoonhoven R, Thurman RG. Dietary glycine prevents increases in hepatocyte proliferation caused by the peroxisome proliferator WY-14,643. Chem Res Toxicol. 1997a;10:1198–1204. [PubMed]
- Rose ML, Germolec DR, Schoonhoven R, Thurman RG. Kupffer cells are causally responsible for the mitogenic effect of peroxisome proliferators. Carcinogenesis. 1997b;18:1453–1456. [PubMed]
- Rose ML, Rivera CA, Bradford BU, Graves LM, Cattley RC, Schoonhoven R, Swenberg JA, Thurman RG. Kupffer cell oxidant production is central to the mechanism of peroxisome proliferators. Carcinogenesis. 1999b;20:27–33. [PubMed]
- Rose ML, Rusyn I, Bojes HK, Belyea J, Cattley RC, Thurman RG. Role of Kupffer cells and oxidants in signaling peroxisome proliferator-induced hepatocyte proliferation. Mutat Res. 2000;448:179–192. [PubMed]
- Rusiecki JA, Holford TR, Zahm SH, Zheng T. Polychlorinated biphenyls and breast cancer risk by combined estrogen and progesterone receptor status. Eur J Epidemiol. 2004;19:793–801. [PubMed]
- Rusyn I, Bradham CA, Cohn L, Schoonhoven R, Swenberg JA, Brenner DA, Thurman RG. Corn oil rapidly activates nuclear factor-kappaB in hepatic Kupffer cells by oxidant-dependent mechanisms. Carcinogenesis. 1999;20:2095–2100. [PubMed]
- Rusyn I, Rose ML, Bojes HK, Thurman RG. Novel role of oxidants in the molecular mechanism of action of peroxisome proliferators. Antioxid Redox Signal. 2000;2:607–621. [PubMed]
- Schramm H, Robertson LW, Oesch F. Differential regulation of hepatic glutathione transferase and glutathione peroxidase activities in the rat. Biochem Pharmacol. 1985;34:3735–3739. [PubMed]
- Sinks T, Steele G, Smith AB, Watkins K, Shults RA. Mortality among workers exposed to polychlorinated biphenyls. Am J Epidemiol. 1992;136:389–398. [PubMed]
- Stemm DN, Robertson LW, Tharappel JC, Spear BT, Lehmler HJ, Glauert HP. Effects of dietary selenium on the hepatic tumor promoting activities of PCBs. Toxicol Sci. 2005;84(1S):303.
- Storelli MM, Storelli A, Marcotrigiano GO. Polychlorinated biphenyls, hexachlorobenzene, hexachlorocyclohexane isomers, and pesticide organochlorine residues in cod-liver oil dietary supplements. J Food Prot. 2004;67:1787–1791. [PubMed]
- Tharappel JC, Lee EY, Robertson LW, Spear BT, Glauert HP. Regulation of cell proliferation, apoptosis, and transcription factor activities during the promotion of liver carcinogenesis by polychlorinated biphenyls. Toxicol Appl Pharmacol. 2002;179:172–184. [PubMed]
- Whysner J, Wang CX. Hepatocellular iron accumulation and increased cell proliferation in polychlorinated biphenyl-exposed Sprague-Dawley rats and the development of hepatocarcinogenesis. Toxicol Sci. 2001;62:36–45. [PubMed]
- Wynford-Thomas D, Williams ED. Use of bromodeoxyuridine for cell kinetic studies in intact animals. Cell Tissue Kinet. 1986;19:179–182. [PubMed]
- Xu YH, Dragan YP, Campbell HA, Pitot HC. STEREO: a program on a PC-Windows 95 platform for recording and evaluating quantitative stereologic investigations of multistage hepatocarcinogenesis in rodents. Comput Methods Programs Biomed. 1998;56:49–63. [PubMed]
- Yamashina S, Ikejima K, Enomoto N, Takei Y, Sato N. Glycine as a therapeutic immuno-nutrient for alcoholic liver disease. Alcohol Clin Exp Res. 2005;29:162S–165S. [PubMed]
|
The publisher's final edited version of this article is available at 
