Dose-response of promotion by polychlorinated biphenyls and chloroform in rat liver foci bioassay

Using the rat liver foci bioassay a dose-response relationship was evaluated for the promoting activity of the ubiquitous and persistent environmental pollutants polychlorinated biphenyls (PCBs) and chloroform. Initiation of liver foci was performed by oral administration of a single dose of 8 mg/kg body wt of diethylnitrosamine to weanling female Sprague-Dawley rats. For polychlorinated biphenyls (PCBs) and chloroform a dose-dependent promoting effect was found. The lowest effective dose of PCBs was 1 mg/kg body wt, given three times a week for 11 concecutive weeks. That of chloroform was 100 mg/kg body wt, administered twice a week for 11 consecutive weeks. The livers were screened for preneoplastic foci 12 weeks after starting the experiments. The amounts of PCBs as well as chloroform normally taken up by humans are greater than a factor of 1000 lower than the effective experimental doses. Thus the risk of human exposure to these chemicals is estimated to be very low. In the case of heavy pollution with PCBs, however, as happened in the Yusho accident (Japan, 1968), the daily intake of PCBs was in the range of the effective doses used in the experiment.Dedicated to Professor Dr. med. Herbert Remmer on the occasion of his 65th birthdaySupported by Grant UBA 106 04 017 from the Umweltbundesamt, Berlin     

Specific binding of polychlorinated biphenyls to rat liver cytosol protein.

Specific binding of polychlorinated biphenyls (PCBs) to rat liver cytosol protein has been detected using the 3H-labeled PCB probe 2,2',4,4',5,5'-hexachlorobiphenyl (6-CB). Binding of 6-CB to cytosol protein is displaced by its non-radioactive congener, is of high affinity (Kd approximately 3 nM) and is saturable (maximal binding capacity Bmax approximately 600 pmol/mg protein). 6-CB binding is not found in liver cytosol of animals fed a PCB-supplemented diet (500 ppm PCB for 5 days). Binding is also in vitro inhibited by high concentrations of triglyceride. PCB congeners such as 3,3',4,4',5-pentachlorobiphenyl as well as the thyroid hormones 3,5,3',5'-tetraiodothyronine and 3,5,3'-triiodothyronine (the latter hormone with an order of magnitude lower affinity) compete for the PCB binding site. On the other hand, a number of biochemically important compounds including the PCB core compound biphenyl and the hormone ligands dexamethasone and estradiol, as well as 2,3,7,8-tetrachlorodibenzo-p-dioxin, do not compete for the 6-CB binding site. The data provide the first evidence of specific binding of unmetabolized PCB congeners to distinct binding sites in rat liver cytosol.     

Tryptophan pyrrolase in rat liver after phenobarbital administration

The administration of phenobarbital increases the activity of tryptophan pyrrolase in the particle-free fraction of rat liver homogenate administration of the drug. The activity of tryptophan pyrrolase remains unaltered after the second day of phenobarbital administration in drinking water given daily until the seventh day. The presence of hematin in the incubation medium increase the specific activity of tryptophan pyrrolase both in the experimental in the control group. The degree of saturation of the enzyme by the heme, however, is higher in the experimental group since its activation in the presence of hematin is lower than in the control group. The presence of phenobarbital in the incubation medium produces a slight inhibitory effect on the activity of tryptophan pyrrolase. Repeated administration of phenobarbital does not increase significantly the induction of tryptophan pyrrolase by hydrocortisone or L-tryptophan.     

Accumulation of methylmercury or polychlorinated biphenyls in in vitro models of rat neuronal tissue

In vivo exposure levels for neurotoxicants are often reported in parts per million (ppm) concentration in tissue, whereas exposure levels in experiments utilizing in vitro models are most commonly reported in micromolar (muM) concentration in the exposure solution. The present experiments sought to determine whether or not in vitro solution concentration was an appropriate dose-metric for comparison to in vivo tissue levels for lipophilic compounds. To do so, the accumulation of the polychlorinated biphenyl (PCB) mixture Aroclor 1254 (A1254) or methylmercury (MeHg) was examined in three commonly utilized in vitro neuronal tissue models: nerve growth factor differentiated pheochromocytoma (PC12) cells, primary cultures of rat neocortical cells, and adult rat hippocampal slices. Tissues were exposed to A1254 (0.65 ppm) or to MeHg (0.0033-0.33 ppm) in serum-free media for 1 or 24 h. Total PCB or mercury accumulation was measured by dual column gas chromatography with electron capture detection or by cold vapor atomic absorption, respectively. PC12 cells accumulated 66.7 and 103.8 ppm PCBs after 1 and 24 h exposure to A1254. Neocortical neurons also accumulated significant concentrations of PCBs, but less so than PC12 cells. After 1 h exposure to 0.65 ppm A1254, slices contained 3.46 and 0.81 ppm PCBs when exposed in a static and perfused system, respectively. After 1 h exposure to 0.0033, 0.033, and 0.33 ppm MeHg, PC12 cells contained 0.3, 2.2, and 17.7 ppm mercury, respectively; after 24 h, PC12 cells contained 0.4, 2.8, and 21.9 ppm. Hippocampal slices accumulated 1.7 and 4.8 ppm mercury after 1 and 3 h exposure to 0.33 ppm MeHg. For comparison, mercury accumulation in rat fetal and pup brain tissue after maternal exposure [0, 0.1, 1.0, or 2.0 mg/kg/day MeHg from gestational day (GD) 6-15] ranged from 0.05 to 7.89 ppm in 0.1 mg/kg dose animals on postnatal day 10 and 2.0 mg/kg dose animals on GD16, respectively. These results demonstrate that accumulation of PCBs and MeHg in vitro is tissue-, time-, and concentration-dependent and indicates that tissue levels rather than exposure concentrations are a more appropriate metric for comparison of in vitro to in vivo effects.     

Effect of polychlorinated biphenyls (PCBs) administration on phospholipid biosynthesis in rat liver

The effect of PCBs or phenobarbital on the biosynthesis of phospholipids in hepatic endoplasmic reticulum of rats was studied by the intraperitoneal injection of [³²P]orthophosphate, [Me?¹⁴ C]choline or [2?³H]glycerol. Significant increases in liver microsomal phospholipid content after the administration of either PCBs or phenobarbital indicated the actual proliferation of endoplasmic reticulum membranes. The rate of both [³²P] and [¹⁴C] incorporations into microsomal choline-containing phospholipids, such as phosphatidylcholine, sphingomyelin and lysophosphatidylcholine, was reduced to one fifth by PCBs administration compared with control animals. The incorporation of [³²P]orthophosphate into phosphatidylethanolamine or other phospholipid classes was less or not affected, respectively, by PCBs administration. The specific inhibitory effect of PCBs on the incorporation into cholinecontaining phospholipids was not observed when [2?³-H]glycerol was used as a precursor. Phenobarbital administration, however, increased significantly the rate of [³²P] incorporation into liver phospholipids, especially phosphatidylcholine. It is suggested that the increase in microsomal phospholipid content by PCBs administration is not due to the stimulation of synthesis but to the inhibition of the catabolism of membrane phospholipids and that the increase in content caused by phenobarbital is due at least in part, to the stimulation of synthesis. The possible site(s) of PCBs-induced inhibition of phospholipid biosynthesis in rat liver is discussed.     

Effects of polychlorinated biphenyls in rat liver: quantitative analysis of enzyme-altered foci.

The promotional effect of various polychlorinated biphenyls and phenobarbital on enzyme-altered lesions in the rat liver was quantified within the framework of the two-stage carcinogenesis model of Moolgavkar and colleagues. The experiment analyzed here followed an initiation-promotion protocol in which female Wistar rats were initiated with diethylnitrosamine (DEN) at 10 mg/kg body wt for 10 days followed by a 8-week period of promoter treatment with various cytochrome P450 isoenzyme inducing and noninducing compounds. This analysis included 4-monochlorobiphenyl, 2,2',4,5'-tetrachlorobiphenyl, 3,3',4,4'-tetrachlorobiphenyl and 3-methylcholanthrene, all administered at 150 mumol/kg body wt, and phenobarbital which was administered continuously in the diet at 0.05% until termination. Animals were killed either 1 or 9 weeks after the end of treatment and their livers were examined for enzyme histological alterations. Focal transections were classified as falling into three phenotypic categories: ATPase dominant, GGT dominant, or ATPase plus GGT (coextensive). A quantitative method was used to analyze the data consisting of the number and sizes of the focal transections. The number of cells altered by the DEN treatment and cell kinetic parameters measuring the promotional effect of the various compounds were estimated. On the basis of these estimates, we computed the number of nonextinct altered foci and their volume fraction as functions of time. We found that foci exhibiting the coextensive phenotype respond most efficiently to promoter treatment, while GGT dominant foci respond weakly to all the promoters with the exception of 3-MC. For phenobarbital, we observed a significant slowing of focal cell proliferation over time.     

Inhibition of dye transfer in rat liver WB cell culture by polychlorinated biphenyls.

In the present investigation the scrape loading/dye transfer assay and microinjection technique are used in order to investigate inhibition of cell-cell communication induced by different polychlorinated biphenyl (PCB) congeners. In these in vitro assays, inhibition of intercellular communication is directly measured as decreased transfer of a fluorescent dye (Lucifer Yellow CH) from donor cells loaded with the dye to surrounding recipient cells. The results show that substitution in the ortho position from the carbon bridge is essential and at least one chloro substituent in ortho position is necessary for the ability to inhibit intercellular communication. The results also suggest that an increase in the number of ortho substituted chlorine atoms in the PCB molecule enhances the ability to inhibit intercellular communication. On the other hand, the total number of substitutions may not be crucial for the ability to inhibit intercellular communication. Our results suggest that PCB-induced down-regulation of intercellular communication is a result of a specific mechanism and not due to unspecific membrane perturbation.     

Biological responses of the rat to polychlorinated biphenyls

A commercial polychlorinated biphenyl mixture (PCBs), Aroclor 1242, was administered to rats po by intubation in order to determine toxic manifestations of acute and subacute ingestion. In addition, the effect of PCBs on hepatic microsomal enzyme systems in rats was evaluated. The oral, 14-day LD50 was determined to be approximately 4.25 g/kg. Major toxic signs observed upon administration of high doses of PCBs included diarrhea, chromodacryorrhea, loss of body weight, unusual stance and gait, lack of response to pain stimuli, and terminal ataxia. Progressive dehydration and CNS depression appeared to be contributing factors in each fatality. Histopathologic alterations were evident only in the liver and kidneys, manifest as foci of sudanophilic vacuolation. Rats maintained on an oral dosage regimen of 100 mg/kg every other day for 3 weeks exhibited similar histopathologic changes, but no overt signs of toxicity. Serum GOT activities were elevated over controls in both the acute and subacute groups. A single ip injection (100 mg/kg) increased liver weight, total hepatic microsomal enzyme activity (measured as hydroxylation of acetanilide and N-demethylation of aminopyrine), and hepatic cytochrome P₄₅₀ and b₅ levels. Hepatic microsomal enzyme activity remained elevated 10 days after a single dose of PCBs, suggesting that PCBs may play an important role in altering biologic responses of mammals subjected to environmental chemical stress.     

Orphan nuclear receptor constitutive active/androstane receptor-mediated alterations in DNA methylation during phenobarbital promotion of liver tumorigenesis.

Altered DNA methylation is an epigenetic mechanism that plays a key role in the carcinogenesis process, and the nongenotoxic rodent hepatocarcinogen phenobarbital (PB) alters the methylation status of DNA in mouse liver. The constitutive active/androstane nuclear receptor (CAR) mediates half of the PB-induced hepatic gene expression changes and it is essential for liver tumor promotion in PB-treated mice. Here, a technique involving methylation-sensitive restriction digestion, arbitrarily primed PCR, and capillary electrophoresis was utilized to detect PB-induced regions of altered DNA methylation (RAMs) in CAR wildtype (WT) mice that are sensitive to promotion by PB and resistant CAR knockout (KO) mice. The CAR WT mice developed preneoplastic lesions after 23 weeks of PB treatment (precancerous) and liver tumors after 32 weeks, while the CAR KO mice did not develop tumors (Y. Yamamoto, et al., 2004, Cancer Res. 64, 7197-7200). Our goal was to discern those RAMs which are playing important roles in tumor formation by comparing the RAMs that form in sensitive and resistant groups of mice. Using this novel approach, 42 unique RAMs were identified in the precancerous as compared to the CAR KO, 23-week PB-treated tissue. Of these 42 RAMs, 14 carried forward to the tumor tissue, and additionally, 104 total unique RAMs were observed in the tumor tissue. These results indicate that there are unique RAMs occurring in the sensitive CAR WT mice and that a portion of these are seen in both the precancerous and tumor tissue. We hypothesize that these unique RAMs may be facilitating the tumorigenesis process, and these data support the view that DNA methylation plays a causative role in PB-induced tumorigenesis.     

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.     

Effects of polychlorinated biphenyls in rat liver: correlation between primary subcellular effects and promoting activity.

In the present study the promoting activity of various PCB and PBB isomers and congeners in rat liver has been studied and compared with a variety of primary xenobiotic-mediated enzymatic changes in this target organ. Female Wistar rats were given diethylnitrosamine (DEN; 10 mg/kg body wt for 10 days) and were subsequently treated once weekly with polychlorinated biphenyls (150 or 15 mumol/kg body wt) for a total of 8 weeks. Additional groups of rats were administered 3,3',4,4'-tetrabromobiphenyl or 3-methylcholanthrene (8 weekly injections of 15 or 150 mumol/kg body wt, respectively) or were given phenobarbital (0.05% in the diet) until the end of the experiment. Reference groups were treated with the various test compounds without prior initiation. One week and 9 weeks after cessation of promoter treatment rats were killed and the volumetric fraction of enzyme-altered foci characterized by changes in adenosine triphosphatase and gamma-glutamyl transpeptidase activity was determined as a means to quantitatively assess the extent of preneoplastic response in this organ. Out of the series of polyhalogenated biphenyls tested, promoting effects were seen with the following compounds: 2,2',4,5'-tetrachlorobiphenyl, 3,3',4,4'-tetrachlorobiphenyl, 2,3,4,4',5-pentachlorobiphenyl, and 3,3',4,4'-tetrabromobiphenyl, whereas no significant effects were obtained with 4-monochlorobiphenyl. In rats not treated with DEN, the two strongly promoting agents 2,3,4,4',5-pentachlorobiphenyl and 3,3',4,4'-tetrachlorobiphenyl also significantly increased the volume fraction of enzyme-altered foci over the respective controls when analyzed at the second time point of investigation. In parallel experiments, induction of liver growth and of microsomal cytochrome P450 content in liver was found to correlate well with the promoting activity of the various xenobiotics, suggesting that these parameters may be used to predict the promoting activity of polyhalogenated biphenyls in a short term assay.     

Rat liver changes after subchronic exposition to polychlorinated biphenyls (PCB)of low chlorine content

Liver changes were studied after subchronic feeding a diet containing 2000 ppm PCB [chlorine content 42% corresponding to trichlorobiphenyls (triCB)]. The products differed in their content of highly chlorinated biphenyls (Cl₅-biphenyl; 5%=triCB-5, 2%=triCB-2 and 0.4%=triCB-0.4). The most striking difference was observed in respect to their porphyrinogenic actions. TriCB-0.4 and triCB-2 caused an increase of porphyrins about 10-fold, whereas the liver porphyrin content of triCB-5 treated rats was 175-fold higher than normal. In all cases of porphyria the porphyrins consisted mainly of uro- and heptacarboxyporphyrin. In contrast to their different porphyrinogenic effects all triCB products caused an equal induction of -aminolaevulinate synthetase, but no increase of -aminolaevulinate dehydratase activity.TriCB-5 was also a stronger inducer than triCB-0.4 and triCB-2 regarding microsomal monoxygenases and UDP-glucuronyltransferase. The quantitative differences of the effect can be correlated to PCB residues in the liver. In triCB-5 fed rats a much higher accumulation of highly chlorinated components could be determined.The histological examination by light and electron microscopy showed no significant differences in the effects caused by all three triCB products. In livers of all rats cell hypertrophy could be observed due to a considerable hyperproliferation of tubular and vesicular SER. SER membranes were tightly packed and sometimes disrupted. Membrane arrays of various size were found in most hepatocytes. The mitochondria were dislocated by these formations and were sometimes swollen. There was neither centrilobular or periportal necrosis nor fibrosis nor fatty degeneration.It may be concluded that PCB of low chlorine content (42%) are much less toxic than highly chlorinated PCB. However, the small amount of highly chlorinated components, present as contaminants in commercial products containing 42% chlorine, may accumulate and exert toxic effects.

---

Zusammenfassung Es wird über Leberveränderungen an Ratten nach subchronischer Fütterung mit 2000 ppm PCB berichtet. Dabei wurden drei Chargen eines technischen Trichlorbiphenyls eingesetzt, die einen unterschiedlichen Gehalt (%) an hochchlorierten Komponenten (d. h.5 Cl) hatten, und zwar: 5% =triCB-5; 2% = triCB-2 und 0,4% = triCB-04. Der auffälligste Wirkungsunterschied der Präparate bestand in den porphyrinogenen Eigenschaften. Während triCB-0,4 und triCB-2 einen Anstieg des Porphyringehaltes der Leber um den Faktor 17 bzw. 7 verursachten, stieg der Gehalt nach triCB-5 175fach höher als normal an. Die Porphyrine bestanden hauptsächlich aus Uroporphyrin und Heptacarboxyporphyrin. Im Gegensatz zu den unterschiedlichen porphyrinogenen Wirkungen bewirkten alle drei triCB-Präparate eine gleich hohe Induktion der -Aminolaevulinatsynthetase. Die -Aminolaevulinatdehydratase erfuhr keine Veränderung ihrer Aktivität.Die mikrosomalen Monoxygenasen und die UDP-Glucuronyltransferase wurden durch alle drei triCB induziert. Die höchsten Werte wurden wieder durch triCB-5 erzielt. Sie korrelieren damit zu den PCB-Rückständen in der Leber, denn nach Fütterung mit triCB-5 kam es zu einer wesentlich höheren Akkumulation der hochchlorierten Komponenten.Bei licht- und elektronenmikroskopischer Untersuchung konnten keine signifikant unterschiedlichen Wirkungen der triCB-Präparate festgestellt werden. Die Lebern aller Tiere zeigten eine erhebliche Zellhypertrophie, die durch die Hyperproliferation des tubulären und vesikulären glatten endoplasmatischen Retikulums hervorgerufen wurde. Die Membranen des Retikulums waren dicht gepackt und manchmal zerrissen. In den meisten Zellen konnten außerdem unterschiedlich große membrane arrays gesehen werden, die zu einer Verdrängung von Mitochondrien führte. Einige Mitochondrien waren geschwollen. Zentrolobuläre oder periportale Nekrosen, Fibrosen oder fettige Degenerationen konnten nicht beobachtet werden.Aus den Untersuchungen kann gefolgert werden, daß PCB-Produkte mit niedrigem Chlorgehalt (42%) wesentlich weniger toxisch sind als solche mit hohem Gehalt. Toxische Effekte können aber dadurch auftreten, daß die in den Handelspräparaten als Verunreinigung enthaltenen hochchlorierten Komponenten akkumulieren.

---

Abbreviations ALA-D -aminolaevulinate dehydratase - ALA-S -aminolaevulinate synthetase - BSP sulfobromophthalein - cyt. cytochrome - diCB commercial PCB mixture (Aroclor^® 1232, Monsanto) of preferably dichlorobiphenyls - hexaCB commercial PCB mixture (Aroclor^® 1260) of preferably hexachlorobiphenyls - tetraCB commercial PCB mixture (Aroclor^® 1248) of preferably tetrachlorobiphenyls - triCB-5, triCB-2, triCB-0.4 PCB mixtures of preferably trichlorobiphenyls containing 5% (2%, 0.4%) highly chlorinated biphenyls ( pentachlorobiphenyls) Some of these results have been reported at the Spring Meeting of the Deutsche Pharmakologische Gesellschaft, Mainz, 1975 (Grote et al., 1975c)This work was carried out within the coordinated research program Polychlorinated Biphenyls in the Environment which was supported by a grant of the German Federal Ministry for Research and Technology