William E. Fahl

Detection of a nafenopin-binding protein in rat liver cytosol associated with the induction of peroxisome proliferation by hypolipidemic compounds

[3H]nafenopin, a known inducer of liver peroxisomal enzymes, was shown to bind to a specific, saturable pool of binding sites in cytosols from rat liver and kidney cortex. Tissue levels of this binding protein (liver greater than kidney cortex; not detectable in myocardium, skeletal muscle) were seen to correlate with the ability of nafenopin to induce peroxisomal enzymes in these organs. Clofibrate and ciprofibrate, which are structurally similar to nafenopin, competitively blocked the specific binding of [3H]nafenopin. Phenobarbital, a non-inducer of peroxisomes, and [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]acetic acid and 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio(N-beta-hydroxyethyl)acetamide, which are structurally unrelated peroxisome proliferators, did not complete for the specific [3H]nafenopin binding sites. The [3H]nafenopin binding protein is proposed as a mediator of the drug-induced increase in peroxisomes and associated peroxisomal enzymes.

Benzo[a]pyrene diol-epoxides different mutagenic efficiency in human and bacteria cells

Monolayer cultures of diploid human fibroblasts and suspensions of S. typhimurium TA100 cells were treated with [3H]-labelled enantiomeric forms of benzo[a]pyrene anti and syn 7,8-dihydrodiol 9,10-epoxides. In both cell types, all of the enantiomers induced the formation of mutant 6-thioguanine (human) or 8-azaguanine-(bacterial)resistant cells. Diol-epoxide-modified nucleosides from human and from bacterial DNA hydrolysates were characterized by HPLC and showed essentially the same adduct species for human and bacterial cells treated with the same enantiomers. There were substantial differences, however, in the efficiency with which structurally-different adduct species were converted to mutant genotypes. In human cells, the mutagenic efficiency (mutation frequency/unit modified DNA) of the respective adduct species (+ anti much greater than -anti = +/- syn) at the hprt locus was exactly the opposite of that seen at a similar gene locus (gpt) in TA100 (-anti = +/- syn greater than + anti). The results suggest that the structural configuration of adducts in genomic DNA is important in determining whether a mutant genotype will result, and likewise, that there are differences in specificity between the human and bacterial systems which process these adduct lesions.

Lack of covalent binding of peroxisome proliferators nafenopin and WY-14 643 to DNA in vivo and in vitro

[3H][2-methyl-2-p-(1,2,3,4-tetrahydro-naphthyl)phenoxy] propionic acid (nafenopin), a hepatocarcinogenic peroxisome proliferator, when administered p.o. to normal intact and partially hepatectomized male F344 rats did not show any significant binding to DNA and RNA, but bound to proteins. The in vitro incubation of [3H]nafenopin and [3H]4-chloro-[6-(2,3-xylidino)pyrimidinylthio]acetic acid (Wy-14643), another peroxisome proliferator, with hepatic microsomes and calf thymus DNA also showed no significant binding of these chemicals to DNA.

The kinetics of benzo(a)pyrene anti-7,8-dihydrodiol 9,10-epoxide formation from benzo(a)pyrene and regulatory membrane effects

Abstract r -7, c -10, t -8, t -9-Tetrahydroxybenzo( a )pyrene (7,10/8,9-tetrol), which is the principal hydrolysis product of r -7, t -8-dihydroxy- t -9,10-oxy-7,8,9,10-tetrahydrobenzo( a )pyrene ( anti -diol-epoxide), was resolved and measured by HPLC in organic extracts of incubations which contained induced rat liver microsomes and BP. Kinetic analyses showed that: (a) following a 5- to 7-min lag period, anti -diol-epoxide formation was linear, and (b) levels of anti -diol-epoxide formed were highly dependent upon the starting BP concentration. anti -Diol-epoxide production increased at starting BP concentrations of 0–12 μ m and decreased in incubations containing 12–25 μ m BP. However, between 25 and 100 μ m BP, anti -diol-epoxide formation was stable at a level representing 65% of the peak production which occurred at a starting BP concentration of 12 μ m . BP oxidation was competitively inhibited by (−)- trans -BP-7,8-dihydrodiol and about five times less effectively by the (+)- trans -BP-7,8-dihydrodiol. The inability of a severalfold excess of BP (25–100 μ m ) to totally inhibit BP-7,8-dihydrodiol oxidation was explained by the presence of a microsomal substrate compartment which was saturated at only 6–8 μ m BP, the remaining BP present as aggregates in the aqueous compartment. Purification of microsomes by Sepharose 2B gel filtration after reaction with [ 3 H]BP also indicated that BP-7,8-dihydrodiol was preferentially concentrated in the microsome compartment leading to a net increase in the ratio of BP-7,8-dihydrodiol to BP in the microsomal compartment, which favored BP-7,8-dihydrodiol oxidation to yield the biologically active anti -diol-epoxide.

Cell-Specific Expression of a Recombinant Rat Glutathione S-Transferase Ya Gene in Transgenic Mice

Transgenic mice have been generated which carry a cDNA encoding the rat Ya isozyme of glutathione S-transferase (GST) under the transcriptional control of the SV40 early region promoter-enhancer. Expression of the GST transgene was highly tissue-specific, with the highest expression detected in the convoluted tubular epithelium of the mouse kidney cortex. GST Ya mRNA abundance in these cells was greater than that found for GST Ya mRNA in normal rat liver. GST Ya protein was observed in the convoluted tubule cells of the founder mouse as well as an F1 offspring. The transmission of the foreign gene was followed for two generations, and an erratic pattern of inheritance was observed. These animals provide a model for the in vivo study of GST modulation of carcinogenesis and drug toxicity.