Larry W. Robertson

Characteristics of polychlorinated biphenyl (PCB) atropisomers

We have prepared and characterized the atropisomers of 3 PCBs, namely 2,2′,3,4,6-penta-[I],2,2′,3,4,4′,6-hexa-[II] and 2,2′,3,3′,4,4′,6,6′-octachlorobiphenyls [III]. On the basis of the rotational stability of these 3 enantiomer pairs, we can predict that, of the 78 PCBs which possess axial chirality, the enantiomers of at least 19 PCBs containing 3 or 4 ortho chlorines would be stable to racemization at room temperature.

A fluorometric assay for quantitating phenol sulfotransferase activities in homogenates of cells and tissues

A new, rapid, and sensitive method for assaying phenol sulfotransferase activity toward 2-naphthol is described. The product 2-naphthyl sulfate is quantitated fluorometrically. Optimal wavelengths for excitation and emission were determined by recording the three-dimensional fluorescence spectra of the substrate and the product. The new method is applicable to crude cell or tissue homogenates as well as to further purified preparations. A comparison to another widely used method is given to point out the advantages provided by the new procedure. In particular, sensitivity and accuracy of both methods are evaluated and the influence of interfering substances on both systems is compared. These results clearly indicate the superiority of the new method.

Perfluorodecanoic acid decreases the enzyme activity and the amount of glutathione S-transferases proteins and mRNAs in vivo

The effects of the anti-wetting agent perfluoro-n-decanoic acid (PFDA) on various glutathione S-transferase (GST) enzyme activities were studied in vitro and in vivo. In addition the effects of PFDA treatment on the amount of some glutathione S-transferase subunits and their corresponding translatable mRNA were studied in vivo. PFDA like some other peroxisome proliferators was a non-competitive inhibitor of several GST enzyme activities in vitro. In vivo PFDA reduced the enzyme activity towards substrates which are indicative for the Ya, Yb1 and Yb2 subunits of GSTs to a larger extent than the enzyme activity towards the substrate indicative for the Yc subunit. Whereas the reduction of GST enzyme activities by other peroxisome proliferators was shown to be caused by an inhibition of the relevant enzymes in vivo, PFDA was found to decrease the GST enzyme activities at least in part by lowering the amount of the various GST subunits in vivo due to a lowered concentration of translatable mRNA coding for these enzymes. In addition PFDA abolished the inducibility of GST mRNAs by phenobarbital. Thus PFDA might be an interesting tool for mechanistic studies of the control of GST expression in the liver.The effects of the anti-wetting agent perfluoro-n-decanoic acid (PFDA) on various glutathione S-transferase (GST) enzyme activities were studied in vitro and in vivo. In addition the effects of PFDA treatment on the amount of some glutathione S-transferase subunits and their corresponding translatable mRNA were studied in vivo. PFDA like some other peroxisome proliferators was a non-competitive inhibitor of several GST enzyme activities in vitro. In vivo PFDA reduced the enzyme activity towards substrates which are indicative for the Ya, Yb1 and Yb2 subunits of GSTs to a larger extent than the enzyme activity towards the substrate indicative for the Yc subunit. Whereas the reduction of GST enzyme activities by other peroxisome proliferators was shown to be caused by an inhibition of the relevant enzymes in vivo, PFDA was found to decrease the GST enzyme activities at least in part by lowering the amount of the various GST subunits in vivo due to a lowered concentration of translatable mRNA coding for these enzymes. In addition PFDA abolished the inducibility of GST mRNAs by phenobarbital. Thus PFDA might be an interesting tool for mechanistic studies of the control of GST expression in the liver.

Absence of lipid peroxidation as determined by ethane exhalation in rats treated with 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD)

The exhalation of ethane is widely used as an indicator of in vivo lipid peroxidation. To test the hypothesis that lipid peroxidative events are involved in the toxicity of 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD), we administered a lethal dose of TCDD (60 μg/kg), IP to male Sprague Dawley rats (160–180 g) and measured by gas chromatography the exhalation of ethane into the atmosphere of a closed all-glass exposure chamber. TCDD-treated rats exhaled only slightly more ethane than control rats at a single time point 7 days following TCDD administration. Since the exhalation of ethane is the net result of the endogenous production of the gas and its metabolic degradation, the latter was quantified by measuring the clearance of exogenous ethane (initial concentration = 100 ppm) introduced to the atmosphere of the exposure chamber. The clearance of ethane in TCDD-treated rats was markedly decreased, reaching a minimum 7 days following TCDD treatment. Apparently, the slight increase in exhaled ethane was due to an inhibition of ethane metabolism caused by TCDD. However, rats obviously intoxicated and having lost considerable body weight might be impaired in their ability to transport ethane. To bypass this problem we injected ethane (0.2 ml) directly into the rats IP. Here also the metabolic clearance in TCDD-treated rats was diminished. In a further experiment, rats treated with dithiocarb at a dose where ethane metabolism was totally inhibited exhaled more ethane than did TCDD-treated rats. It is therefore concluded that the slight increase in ethane exhalation following a lethal dose of TCDD is due to a partial inhibition of ethane metabolism and that there is no net increase in ethane production due to lipid peroxidation. Indeed when TCDD-treated rats were administered Fe++, a well-known initiator of lipid peroxidation, they were less competent to carry out lipid peroxidation than rats treated with Fe++alone.

Selective induction of bilirbuin UDP-glucuronosyl-transferase by perfluorodecanoic acid

Differential effects of perfluorodecanoic acid (PFDA) on rat liver UDP-glucuronosyltransferase isoenzymes have been observed after a single i.p. administration of the compound to young male Sprague-Dawley rats. (1) Bilirubin glucuronidation was induced 2-fold. The induced state was stable for at least 3 weeks. (2) Glucuronidation of 1-naphthol, morphine and testosterone was decreased to half of the control values. These decreases were maximal after 12 days but all three activities returned to normal levels after 3 weeks. (3) Immunoblotting experiments indicated that the differential effects of PFDA on UDP-glucuronosyltransferase activities were due to modulation of enzyme protein concentrations rather than activation/inactivation mechanisms. With respect to its influence on UDP-glucuronosyltransferase isoenzymes, PFDA may be classified as a clofibrate-type inducer. The persistence of the induction after a single application however is unique among peroxisome proliferators and therefore PFDA may be a useful, elective inducer of bilirubin glucuronidation.

Differential induction of cytochrome P-450 by the enantiomers of trans-stilbene oxide

Optically pure (+)- and (-)-trans-stilbene oxide (TSO) enantiomers were administered to immature male Sprague-Dawley rats. (+)-TSO was the more potent inducer of liver microsomal cytochrome P-450-dependent monooxygenases. The greater potency of (+)-TSO may be explained on the basis of stereoselective metabolism since a far greater concentration of TSO was found in liver microsomes of (+)-TSO-treated rats. Furthermore, of the enzymes known to metabolize TSO, cytosolic epoxide hydrolase turned over the (-)-TSO enantiomer at a faster rate, consistent with the greater persistence of the (+)-enantiomer. Although this report is of chiral effects in potency of enzyme induction, stereoselective metabolism (i.e. disposition) rather than inherent structural characteristics (recognition) may be responsible for these effects.

Cis- and trans-1,2-diphenylaziridines: induction of xenobiotic-metabolizing enzymes in rat liver and mutagenicity in Salmonella typhimurium

Abstracttrans-Stilbene imine (trans-1,2-diphenylaziridine) is the nitrogen analog of trans-stilbene oxide, a potent inducer of several microsomal and cytosolic xenobiotic-metabolizing enzymes. Although the acute toxicity of cis- and trans-stilbene imines prevents their application at the usual dose for trans-stilbene oxide (400 mg/kg/day), it is apparent that the imines nevertheless potently induce several xenobiotic-metabolizing enzymes in rat liver. The IP administration of trans-stilbene imine resulted in statistically significant increases in the activities of aminopyrine N-demethylase, microsomal epoxide hydrolase, glutathione transferase (toward 1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4-nitrobenzene and Δ5-androstene-3,17-dione) and UDP-glucuronosyltransferase (toward testoster-one). cis-Stilbene imine was less potent in inducing these activities. Although trans-stilbene imine (total dose = 400 mg/kg) was more potent than trans-stilbene oxide (total dose = 1200 mg/kg) in inducing the activities of glutathione transferase (toward 1-chloro-2,4-dinitrobenzene) and UDP-glucuronosyltransferase (toward testosterone), both compounds belong to the class of substances which are more potent inducers of conjugating (phase II) enzymes.Because of their structural similarity with K-region arene imines which are potent mutagens, cis-stilbene imine and trans-stilbene imine were investigated for mutagenicity (reversion of his− strains of Salmonella typhimurium). cis-Stilbene imine and trans-stilbene imine were direct mutagens in the strain TA100. This result, and the finding that acenaphthene 1,2-imine efficiently reverts various strains of Salmonella typhimurium, demonstrates that not only K-region arene imines, but also other aziridines substituted at the two carbons with aromatic moieties, are mutagenic.

Effects of congeneric polychlorinated biphenyls on liver and kidney retinoid levels

Abstract Liver and kidney retinol and retinyl palmitate levels were studied in male Sprague Dawley rats receiving a single ip injection of one of several polychlorinated biphenyls (PCBs) or DDT. Only the administration of 3,3′,4,4′-tetrachlorobiphenyl resulted in progressively lowered liver vitamin A levels (to 40 % of control values by day 7). During this time, kidney total vitamin A content increased 3 fold (due primarily to increased retinol content), but this increase was only equal to approximately 1 40 of total vitamin A which had disappeared from the liver. The activity of retinyl palmitate hydrolase, the key enzyme in the hydrolysis of hepatic retinyl palmitate, was not increased, indicating that this enzyme is probably not involved in the depletion of liver vitamin A stores by 3,3′-4,4′-tetrachlorobiphenyl.