Garold S. Yost

Decreased pneumotoxicity of deuterated 3-methylindole: Bioactivation requires methyl CH bond breakage☆

The bioactivation of the pulmonary toxin 3-methylindole has been postulated to proceed via the formation of an imine methide. To test this hypothesis, the toxicity in mice of 3-methylindole has been compared to the toxicity of its perdeuteromethyl analog. Deuteration of the methyl group should slow the rate of production of the corresponding imine methide and diminish the toxicity of deutero-3-methylindole, if C-H bond breakage occurs prior to or during the rate-determining step. In agreement with this hypothesis, deutero-3-methylindole was synthesized and was shown to be significantly less toxic (LD50 735 mg/kg) than 3-methylindole (LD50 578 mg/kg). Both compounds produced the same lesion at the LD50 dose, bronchiolar damage and mild alveolar edema, indicating that deuteration of 3-methylindole did not change the pathologic process. However, at a much lower dose (25 mg/kg), 3-methylindole produced a mild bronchiolar lesion whereas deutero-3-methylindole did not damage lung tissue. Additionally, administration of deutero-3-methylindole caused less pulmonary edema compared to 3-methylindole, as assessed by increased wet lung weights. Finally, the depletion of pulmonary glutathione by deutero-3-methylindole was considerably slower than depletion by 3-methylindole. The electrophilic imine methide has been postulated to be the intermediate which binds with and depletes glutathione. Therefore, the evidence presented here supports the involvement of an imine methide as the primary reactive intermediate in 3-methylindole-mediated pneumotoxicity.

Absorption of 3-nitropropanol and 3-nitropropionic acid from the digestive system of sheep

When 3-nitropropanol (NPOH) was injected into the rumen (30 mg/kg), abomasum (10 mg/kg) or small intestine (10 mg/kg) of sheep, it was rapidly absorbed and converted to 3-nitropropionic acid (NPA). The reticulo-rumen was the major site of absorption for the miserotoxin aglycone but the abomasum and the small intestine also had the capacity to absorb NPOH. When NPA was injected into different regions of the alimentary tract, the reticulo-rumen was also the major site of absorption. Absorption of NPA or NPOH from the small intestine was much more rapid than from the abomasum. Plasma levels of NPA and inorganic nitrite were higher after dosing with NPOH than with NPA indicating a more rapid rate of uptake of the aglycone.

Adducts of 3-methylindole and glutathione: Species differences in organ-selective bioactivation

Lung and liver microsomes of several species were evaluated for potential to form activated metabolites of 3-methylindole (3MI). Microsomes were incubated with [14C]3MI and glutathione (GSH). Electrophilic 3MI metabolites were trapped and quantitated as GSH adducts by HPLC, and by determining the amounts of activated intermediates which became covalently bound to microsomal protein. The highest rates of 3MI-GSH adduct formation by the lung were detected in microsomes of the goat, followed in decreasing order by pulmonary microsomes from the horse, monkey, mouse, and rat, respectively. In contrast, hepatic 3MI-GSH adduct production was highest in microsomes from the rat, followed by mouse, monkey, goat, and horse microsomes, respectively. These results suggest that the species and organ-selective toxicity of 3MI are primarily caused by differences in rates of oxidative metabolism of 3MI to an electrophilic intermediate.

Glutathione adduct formation with microsomally activated metabolites of the pulmonary alkylating and cytotoxic agent, 3-methylindole☆

Incubations with goat lung and liver microsomes were conducted to trap with exogenous glutathione (GSH) the electrophilic intermediate produced via cytochrome P-450-dependent metabolic activation of 3-methylindole (3MI). Microsomal incubation mixtures with [14C]3MI, a NADPH-generating system, and [3H]GSH produced a dual-labeled adduct which was isolated by reverse-phase high-performance liquid chromatography. Reactive 3MI intermediates were also trapped with cysteine. Adduct formation increased in proportion to the concentration of either thiol. Covalent binding of activated 3MI metabolites to microsomal protein was inversely related to adduct production. There were both qualitative and quantitative differences in the formation of GSH adducts by lung and liver microsomes. In the presence of 2 mM GSH, the adduct was produced at a rate of 1.8 nmol/mg protein/min by lung microsomes but only at 0.1 nmol/mg protein/min by hepatic microsomes. The addition of cytosolic fractions containing glutathione S-transferase activity increased GSH adduct formation by approximately 30%. These results support the view that electrophilic 3MI intermediates are trapped by conjugation with GSH, and that organ-selective toxicity is primarily due to much faster rates of cytochrome P-450 oxidation of 3MI in the lung than in the liver.

Conversion of 3-nitropropanol (miserotoxin aglycone) to 3-nitropropionic acid in cattle and sheep

The metabolism of intravenously administered 3-nitropropanol (miserotoxin aglycone) was examined in cattle and sheep using high-pressure liquid chromatography (HPLC) to determine nitrocompounds in plasma. 3-Nitropropanol (NPOH) showed a rapid rate of decay and simultaneously, 3-nitropropionic acid (NPA) was detected at comparable levels in plasma. In animals dosed with NPOH, the observed NPA decayed at a slower rate than NPOH. Nitrite levels in plasma were more closely related to NPA than to NPOH. Therefore, metabolism of NPOH is linked to NPA and this could provide a common basis for the toxicity of these compounds in cattle and sheep.

Effect of alcohol and aldehyde dehydrogenase inhibitors on the toxicity of 3-nitropropanol in rats

The nitrocompounds 3-nitropropanol (NPOH) and 3-nitropropionic acid (NPA) were shown to be equally toxic when injected intraperitoneally into male Wistar rats. The LD50 for NPOH was 0.58 mmol/kg and for NPA it was 0.56 mmol/kg. NPOH was rapidly metabolized to NPA but this conversion was suppressed by prior administration of ethanol or 4-methylpyrazole to inhibit alcohol dehydrogenase. Administration of ethanol or 4-methylpyrazole before NPOH protected rats from intoxication. However, if the alcohol dehydrogenase inhibitors were given after the nitroalcohol, toxicity still occurred. Administration of the aldehyde dehydrogenase inhibitor diethyldithiocarbamic acid had little effect on the conversion of NPOH to NPA and did not alter the toxicity of NPOH. It was concluded that NPOH and NPA are equally toxic to rats but that NPOH is toxic due to its being rapidly converted to NPA.

Substrate-selective induction of rabbit hepatic UDP-glucuronyltransferases by ethanol and other xenobiotics.

Male New Zealand white rabbits were treated with various inducers of hepatic metabolism enzymes to characterize the induction of UDP-glucuronyltransferase (UDP-GT) enzymes. Rabbits were pretreated with phenobarbital, 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT), 3-methylcholanthrene, beta-naphthoflavone, Aroclor 1254, ethanol, trans-stilbene oxide, pregnenolone-16 alpha-carbonitrile, or clofibric acid. Hepatic microsomes from treated and control animals were incubated with the GT1-type substrates, p-nitrophenol and 1-naphthol; the GT2-type substrate, morphine; and the steroid substrate, estrone. Compared to the rat, the rabbit was particularly resistant to UDP-GT induction. Ethanol was the most potent inducer for both GT1 and GT2 activities, but it failed to induce steroid (estrone, estradiol, and testosterone) UDP-GT activities. Ethanol pretreatment increased oxazepam-GT but it decreased bilirubin-GT activity. 3-Methylcholanthrene (3MC) and beta-naphthoflavone (BNF) are the prototypic GT1 inducers in the rat, but 3MC caused no induction of GT1 activity and BNF caused induction of both GT1 and GT2 activities in the rabbit. None of the xenobiotic pretreatments increased the hepatic microsomal glucuronidation of estrone. These results demonstrate that the induction of UDP-GT activities, and the use of this phenomenon to classify UDP-GT forms, is somewhat species-specific and cannot necessarily be extrapolated from rats to other species. In addition, the substrate selectivity of ethanol-induced microsomal UDP-GT was established.

Ethanol as an inducer of UDP-glucuronyltransferase: A comparison with phenobarbital and 3-methylcholanthrene induction in rabbit hepatic microsomes

In this report we have examined the ability of ethanol to induce UDP-glucuronyltransferase activity in male rabbits using p-nitrophenol as substrate. The rabbit was found to be an excellent species for studies of ethanol induction since almost 3-fold increases in activity were observed relative to controls. Ethanol induction of p-nitrophenol UDP-glucuronyltransferase activity was even greater than induction by 3-methylcholanthrene, the prototypic inducer of this type of activity. Thus, the rabbit shows promise for studies of UDP-glucuronyltransferase isozymes that are induced by chronic ethanol consumption.

Electrophilic metabolites of 3-methylindole as toxic intermediates in pulmonary oedema

[methyl-14C]-3-Methylindole (3MI) was incubated with goat-lung microsomes, an NADPH-generating system and glutathione. An adduct between an oxidative metabolite of 3MI and glutathione was formed only when the complete system was employed. The adduct, which was detected by u.v. absorbance and scintillation counting of h.p.l.c. fractions, was purified to homogeneity by reverse-phase h.p.l.c. The ability of 3MI to bind to microsomal protein was reduced to 52% and 46% of controls when 2 mM and 4 mM glutathione, respectively, were included in the incubations. These results suggest the involvement of an electrophilic metabolite as the toxic intermediate in 3MI-mediated pulmonary oedema.

Procarbazine spermatogenesis toxicity: Deuterium isotope effects point to regioselective metabolism in mice

Procarbazine was shown to decrease spermatogenesis in male mice in a dose-dependent manner. Significant decreases (44% of controls) in spermatogenesis were observed when a dose of 400 mg/kg was administered 18 days prior to determination of sperm count. Procarbazine caused no significant acute spermatocidal activity in vivo. Procarbazine-associated decreases in spermatogenesis were thus used as an index of toxicity to developing spermatid cells. Procarbazine analogs were synthesized that had deuterium substituted for hydrogen at the benzylic position, N-isopropyl-alpha-(2-methylhydrazino)-p-[alpha, alpha-2H2]toluamide (d2-procarbazine), or at the methyl position, N-isopropyl-alpha-(2-[alpha, alpha, alpha-2H3]methylhydrazino)-p-toluamide (d3-procarbazine). Spermatogenesis decreases caused by d3-procarbazine were essentially the same as with procarbazine in mice (66% of controls at a dose of 200 mg/kg), but d2-procarbazine was nontoxic to developing sperm cells (99% of control at a dose of 200 mg/kg). The decrease in toxicity caused by deuterium substitution at the benzylic position, coupled with the absence of an effect with the methyl-labeled analog, indicate the requirement for regioselective oxidative metabolism of procarbazine at the benzylic position prior to the toxic event.