Dieter Schrenk

Potency of mixtures of polychlorinated biphenyls as inducers of dioxin receptor-regulated CYP1A activity in rat hepatocytes and H4IIE cells

Among the polychlorinated biphenyls (PCBs), a family of widespread environmental pollutants, the most toxic non-ortho-substituted coplanar (non-ortho coplanar) congeners are thought to act as strong dioxin (aryl hydrocarbon) receptor agonists leading to adverse effects, such as body weight loss, immunosuppression, thymic atrophy, hepatotoxicity, tumor promotion, and disturbances of steroid hormone action. Since PCBs are present in environmental and tissue samples as complex mixtures, we investigated the possible interaction of non-ortho coplanar congeners with other major PCBs, which are less active or inactive as dioxin receptor agonists. As a parameter for dioxin receptor activation, induction of CYP1A-catalyzed 7-ethoxyresorufin O-deethylase (EROD) was determined in rat hepatocytes in primary culture and in the rat hepatoma cell line H4IIE. In rat hepatocytes, individual EC50-values and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) equivalency factors (TEFs) for the non-ortho and mono-ortho coplanar PCBs 126, 169, 105, 118 and 156, were in good agreement with published data from in vivo experiments, while in H4IIE cells coincidence was lower. However, in both cell systems TEFs for PCB 77 were significantly higher than reported from experiments in rats. In an approximately equipotent mixture the six potent PCB congeners showed perfect additive behaviour in both cell systems. In contrast, addition of a tenfold surplus of abundant mono- and di-ortho PCBs (28, 52, 101, 138, 153 and 180) led to an almost threefold higher TEF than predicted. This finding suggests a moderate synergistic enhancement of the inducing potency of potent PCBs by less potent congeners, present in abundance in environmental and tissue samples.

ASSESSMENT OF BIOLOGICAL ACTIVITIES OF MIXTURES OF POLYCHLORINATED DIBENZO-P-DIOXINS : COMPARISON BETWEEN DEFINED MIXTURES AND THEIR CONSTITUENTS

As a first step to assess biological activities of complex mixtures of polychlorinated dibenzo-p-dioxins (PCDDs), induction of 7-ethoxyresorufin O-deethylase (EROD) by defined mixtures and their constituents has been analysed in vitro. Two cell systems have been compared: primary hepatocyte cultures and hepatoma H4IIE cells. EC50 values of PCDDs were compared with that of the most potent compound, 2,3,7,8-Cl4DD (2,3,7,8-tetrachlorodibenzo-p-dioxin) and expressed as 2,3,7,8-Cl4DD equivalents (TEs). TEs for three defined mixtures containing up to 49 PCDDs could be predicted from the sum of TEs for the 2,3,7,8-substituted congeners. Efficacies (maximal enzyme induction) of less potent PCDDs (1,2,3,4-Cl4DD, Cl8DD and of a mixture containing 86% Cl8DD and of benz(a)anthracene were lower in hepatocytes (by 33%) and in H4IIE cells (by 50%). The results suggest that biological activities of complex PCDD mixtures are largely due to additive effects of their 2,3,7,8-substituted consituents.

Assessment of biological activities of mixtures of polychlorinated dibenzo-p-dioxins (PCDDs) and their constituents in human HepG2 cells

Dose-response curves of the induction of P4501A1-dependent 7-ethoxyresorufin O-deethylase (EROD) were analyzed in human hepatoma HepG2 cells treated with defined mixtures of polychlorinated dibenzop-dioxins (PCDDs) and their 2,3,7,8-substituted constituents, similar to previous studies with rat hepatocytes and H4IIE cells (Schrenk et al. 1991). PCDDs appear to act less potent in human HepG2 cells in comparison with rat cells. For example, EC50 values of 2,3,7,8-Cl4DD were 8-fold and 19-fold higher than in rat H4IIE cells and hepatocytes, respectively. EC50 values of PCDDs were compared with that of 2,3,7,8-Cl4DD and expressed as 2,3,7,8-Cl4DD equivalents (TEs). Although the rank order of PCDD potencies was similar, TEs for some PCDDs (1,2,3,7,8-Cl5 DD; TE=0.75 and 1,2,3,4,7,8-Cl6DD; TE=0.61) were found to be higher than in the rat system. In contrast to rat cells no significant induction of EROD could be detected with Cl8DD in HepG2 cells up to its limit of solubility. Experimentally determined TEs of PCDD mixtures containing 49 constituents were found to be largely due to additive effects of their 2,3,7,8-substituted constituents.

DRUG METABOLIZING ENZYME ACTIVITIES IN RAT LIVER EPITHELIAL CELL LINES, HEPATOCYTES AND BILE DUCT CELLS

P450-dependent mono-oxygenase and conjugating enzyme activities were studied in rat liver epithelial cells (RLEs) and compared to those in hepatocytes and bile duct cells. Various RLE cell lines were investigated since (a) they are suspected to be derived from cells in the lineage from putative pluripotent stem cells to either hepatocytes or bile duct cells, and (b) they may represent targets of chemical carcinogens. Despite considerable variation between lines, common features were recognized. P450-dependent monooxygenase activities (7-ethoxyresorufin O-deethylase and 7-ethoxycoumarin O-deethylase) were undetectable in all RLEs and bile duct cells, and were uninducible by benz(a)anthracene. In contrast, glucuronosyltransferase (GT), sulfotransferase and GSH transferase activities were clearly detectable. Conjugating enzyme activities increased until confluency of the cell cultures was reached. Under the latter conditions, GT activities towards 4-methylumbelliferone or benzo(a)pyrene-3,6-quinol (substrates of a 3-methylcholanthrene-inducible phenol GT) were similar to those found in hepatocytes or bile duct cells. Using a selective cDNA probe, phenol GT mRNA was clearly detectable in RLE1. In contrast, GT activity towards 4-hydroxybiphenyl was much lower than in hepatocytes or bile duct cells (0.04- and 0.03-fold). Sulfotransferase and GSH transferase activities were also roughly comparable to those found in hepatocytes and in bile duct cells. The results suggest that RLEs and bile duct cells exhibit both high conjugating enzyme activities and a lack of P450-dependent mono-oxygenase activities, a pattern resembling the toxin-resistance phenotype found in putative preneoplastic hepatocyte foci and nodules.

CYP1A1-inducing potency in H4IIE cells and chemical composition of technical mixtures of polychlorinated biphenyls

Polychlorinated biphenyls (PCBs) are present in environmental and tissue samples as complex mixtures of dioxin-like and non-dioxin-like congeners. Induction of cytochrome (CYP) P4501A1-catalyzed 7-ethoxyresorufin-O-deethylase (EROD) activity in H4IIE hepatoma cells is widely used as a simple in vitro bioassay for the dioxin receptor-mediated biological action of dioxin-like agonists. Since the results of the assay may be influenced indirectly by abundant non-dioxin-like PCBs, its application to the bioanalysis of complex PCB mixtures was studied. In the PCB mixtures Arochlor 1254 and Clophen A50, potent dioxin-like non-ortho PCBs and polychlorinated dibenzofurans (PCDFs) were found in minor amounts. However, the non-ortho PCBs accounted for most of the overall 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) equivalents based on EROD induction (EROD-TEQs). A comparison with a pattern of toxic equivalents (TEQs) based on toxic equivalency factors (I-TEFs) recently suggested in an international report revealed a much higher relative impact of mono-ortho PCBs on I-TEQs than on EROD-TEQs while total EROD-TEQs approximately coincided with total I-TEQs. It is concluded that the H4IIE bioassay is useful to assess total I-TEQs but does not reflect the individual contributions of PCB subgroups because of a higher evaluation of mono-ortho and di-ortho PCBs by I-TEFs. Based on individual EROD-TEFs, slightly higher mean EROD-TEQs than those calculated by assuming additive behaviour of single PCBs were obtained. This finding suggests a minor synergistic influence of non-dioxin-like PCBs on the inducing potency of dioxin-like agonists in the H4IIE bioassay.

Tryptanthrins: A novel class of agonists of the aryl hydrocarbon receptor☆

2,3,7,8-Tetrachlorodibenzo-p-dioxin and related environmental pollutants exert most of their adverse effects such as immunosuppression, induction of endocrine dysfunction, tumor promotion, and teratogenicity via the aryl hydrocarbon or dioxin receptor. While most potent agonists of the aryl hydrocarbon receptor are of synthetic origin, an increasing number of natural compounds are now recognized as receptor agonists. Our findings demonstrated that some tryptanthrin derivatives biosynthesized in incubations of Candida lipolytica with tryptophan and anthranilic acid or its derivatives were agonists of the aryl hydrocarbon receptor. The biosynthetic products 8-methyltryptanthrin, 8-chlorotryptanthrin, and 8-bromotryptanthrin induced cytochrome P4501A1 mRNA and protein in rat hepatocytes in primary culture, characteristic features of aryl hydrocarbon receptor agonists. Log-probit analysis of the catalytic activity of cytochrome P4501A1, 7-ethoxyresorufin O-deethylase (EROD), revealed EC50 induction values of 1.7, 0.25, and 0.17 microM for 8-methyltryptanthrin, 8-chlorotryptanthrin, and 8-bromotryptanthrin, respectively. Interestingly, the nonsubstituted tryptanthrin molecule, biosynthesized from the common physiological precursors tryptophan and anthranilic acid, was also active as an inducer. The specificity of the inducing effect of tryptanthrins was demonstrated in gel retardation experiments in Hepa-1 mouse hepatoma cells, showing the characteristic interaction of the activated aryl hydrocarbon receptor with an oligonucleotide containing a xenobiotic-responsive element. It is suggested that the receptor may be part of a defense system protecting higher organisms from secondary metabolites formed by the microflora of the host or its environment.

Multidrug resistance gene expression in rodents and rodent hepatocytes treated with mitoxantrone.

Overexpression of P-glycoprotein in tumor cells can represent a severe drawback for cancer chemotherapy. P-glycoprotein acts as an efflux transporter for a variety of chemotherapeutic agents. It is encoded by multidrug resistance (mdr) genes of the subfamily 1 in humans (MDR1) and rodents (mdr1a and 1b). Because mdr1 gene expression is inducible in cultured rat hepatocytes and in rat liver with chemical carcinogens such as 2-acetylaminofluorene or aflatoxin B1, which form DNA-binding electrophiles during their metabolism, we investigated whether the DNA-damaging chemotherapeutic drug mitoxantrone may induce multidrug resistance in rodents and in hepatocytes in primary culture. In H4IIE rat hepatoma cells stably transfected with a luciferase construct containing the rat mdr1b promoter, mitoxantrone caused a concentration-dependent increase in promoter activity. Mdr1 gene expression in cultured rat hepatocytes was enhanced at mitoxantrone concentrations greater than or equal to 0.1 microM and in mouse hepatocytes at 5 microM. In hepatocytes from both species, a correlation was found between mdr1 induction and the inhibition of protein synthesis. In vivo, mitoxantrone was a very powerful inducer of mdr1 gene expression in rat liver and small intestine. In rat kidney, induction of mRNA was lower, and a marginal effect was seen in lung. In contrast with rats, no significant induction of mdr1 gene expression was obtained in mouse liver. Probably as a consequence of inhibition of protein synthesis, mitoxantrone did not lead to a pronounced elevation of P-glycoprotein levels in rat liver and kidney.

Polyfluorinated dibenzodioxins and dibenzofurans--synthesis, analysis, formation and toxicology.

The 75 congeners of the polyfluorinated dibenzodioxins (PFDDs) and about half of the 135 polyfluorinated dibenzofurans (PFDFs) have been synthesized by pyrolysis of fluorophenols and fluorobenzenes. The individual congeners were characterized by GC/MS. 2,3,7,8-TFDD was also characterized by 1H-, 13C- and 19F-NMR spectroscopy. The retention behaviour of PFDDs and PFDFs during gaschromatographic separation is entirely different from that of PCDDs/PCDFs or PBDDs/PBDFs. The PFDDs/PFDFs elute earlier than the PCDDs/PCDFs and the order of the elution is not governed by the degree of substitution, O8FDD eluting e.g. much earlier than the M1FDDs. A preliminary toxicological evaluation of 2,3,7,8-TFDD was carried out. The elimination of 2,3,7,8-TFDD from mice after a single i.p. injection is biphasic with a very rapid elimination half-life of 5 minutes and a slower phase of 165 minutes. This means a dramatically reduced half-life compared to 2,3,7,8-TCDD with 8.5 d. In liver the TFDD level reaches a maximum 30 minutes after injection and also declined in a biphasic manner. In rat hepatocytes a primary culture induction of CYP4501A1-catalyzed EROD activity could be demonstrated, indicating that 2,3,7,8-TFDD activates the dioxin receptor. In rat hepatocyte cultures similar EC50 values were found for 2,3,7,8-TCDD and 2,3,7,8-TFDD. So far no de novo synthesis of PFDD/PFDF could be detected under conditions were PCDDs/PCDFs are formed. Also, formation of PFDDs/PFDFs could not be detected during thermal treatment of fluorotrichloromethane or Teflon.

Metabolic degradation, inducing potency, and metabolites of fluorinated and chlorinated-fluorinated dibenzodioxins and dibenzofurans

The metabolic degradation of fluorinated, chlorinated-fluorinated and chlorinated congeners was measured in liver homogenate of NMRI mice. While in the time period between 0 and 240 min no degradation of the 2,3,7,8-TCDD/TCDF could be detected, for all fluorinated congeners a perceptible degradation was found, even for the 2,3,7,8-TFDD. Stepwise chlorination of the 2,3,7,8-fluorinated congeners leads to a decrease of the degradation rate. In the EROD test, the exchange of chloro- with fluorosubstituents in the 2,3,7,8-TCDF leads to a decrease of induction potency. 3,7-Dichloro-2,8-difluorodibenzofuran was about 1/1000th as potent as 2,3,7,8-TCDF, while 2,3,7,8-TFDF was complete inactive. Comparison of the metabolic rates of different TCDD with those of the analogous TFDD demonstrates that the order of enzymatic degradation of different TCDD and the analogous TFDD is identical. The TFDD are degraded slightly faster than the corresponding TCDD. Surprisingly 1,4,6,9-TXDD showed the second slowest metabolic rate of the fluorinated and chlorinated TXDD after 2,3,7,8-TXDD although none of the 2,3,7,8-positions were substituted. Judging from 2,3,7,8-TFDD and 1,7-dichloro-2,8-difluorodibenzofuran the metabolic pathway of fluorinated and chlorinated-fluorinated congeners seem to be comparable to the chlorinated congeners.

Induction of hepatic P450-dependent monooxygenase in feral mice from a PCDD/PCDF-contaminated area

Abstract Increased PCDD/PCDF levels were detectable in liver of feral mice (Microtus arvalis) living in PCDD/PCDF contaminated areas (up to 27800 ng TE/kg soil) of a former cable pyrolysis plant, in comparison with animals from a control area. The pattern of PCDD/PCDF congeners showed marked differences between soil and liver. Hepatic 7-ethoxyresorufin O-deethylase (EROD; a functional parameter for PCDD/PCDF-inducible cytochrome P450IA1) was increased in feral mice in relation to the degree of soil contamination with PCDD/PCDF. The data suggest that hepatic EROD activity is a useful bioindicator of the exposure of wild mammalian populations with PCDD/PCDF.