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An 18-year-old girl and her boyfriend were sprayed with toxic chemicals from the left front door of their parked car. The girl died of intoxication about 4 h later. The main findings of postmortem examination were spotty discolorations on her skin and severe pulmonary edema. The unknown chemical was identified as chloropicrin by gas-liquid chromatography and gas chromatography-mass spectrometry of her lung and on samples from the car's console box, cassette tape case, and floor mat, all of which were wet with chemicals. The content of chloropicrin in her lung was calculated at 1.6 ng/g of wet weight.

The biosynthesis of ubiquinone was studied in an isolated perfused beating heart preparation from adult male rats to determine rate-limiting steps in the biosynthetic pathway. The isolated heart could incorporate p-hydroxy[U-14C]benzoate into ubiquinones (ubiquinone-9 and -10) and two other lipids which were identified as 3-nonaprenyl 4-hydroxybenzoate and 3-decaprenyl 4-hydroxybenzoate. No other lipids could be detected. Addition of unlabeled mevalonolactone to the perfusate stimulated the rate of incorporation of p-hydroxy[U-14C]benzoate into 3-nonaprenyl 4-hydroxybenzoate and 3-decaprenyl 4-hydroxybenzoate. The level of radioactivity in these intermediates was much greater than that in ubiquinone-9 and -10. These results show that in the intact heart there is a large excess capacity to form postmevalonate isoprenoid precursors of ubiquinone and suggest a possible regulatory step at the premevalonate level. Moreover, the accumulation of prenylated derivatives of 4-hydroxybenzoic acid indicates further rate limitation at one or more of the subsequent steps in conversion of these intermediates to ubiquinone.

The effects of fenitrothion and methylparathion on the activities of cholinesterase and hepatic microsomal monooxygenases were investigated and Compared following a single or repeated oral administration of an equimolar and low dose of the insecticides. The activities of cholinesterase in brain and plasma were inhibited equally by the repetitive administration of both insecticides. Aminopyrine N-demethylase activity and cyt. P-450 content were not affected under the same experimental conditions. However, methylparathion, when given repeatedly, inhibited the dearylation and desulfuration of its own. The results may indicate that low dose continuous exposure to methylparathion could cause the depression of its own metabolism in rat.

The activities of mitochondrial type A and B monoamine oxidase were determined in the liver of rats fed a diet containing 2-acetylaminofluorene (AAF). Three days after the initiation of AAF-feeding, there was a significant decrease of type B monoamine oxidase activity without affect on type A enzyme. The decreased activity of type B monoamine oxidase, which reached a minimum after three weeks, was sustained for as long as AAF-feeding was continued. Sex-related difference in response to AAF was seen in the rat with respect to the onset and the intensity of the decreased type B monoamine oxidase activity, male rats being more sensitive to the carcinogen than female rats. In contrast to the in vivo effect, AAF showed a potent inhibitory effect on type A monoamine oxidase, rather than on type B enzyme, when added in vitro. The p I 50 values were estimated to be 7.5 against type A monoamine oxidase and 4.1 against type B enzyme, respectively. The in vitro inhibition of both types of monoamine oxidase by AAF was competitive. The K i values for AAF were calculated to be 9.51 · 10 −9 M for type A monoamine oxidase and 1.30 · 10 −5 M for type B enzyme, respectively. In accordance with the potent inhibitory effect of AAF on type A monoamine oxidase in vitro, a single administration of the carcinogen, at a dose of 50 mg/kg, resulted in a marked and temporal decrease of the enzyme activity in the mitochondria of male rat liver. Recovery of the decreased type B monoamine oxidase activity was slow, and the enzyme activity did not return to control levels, even if rats were fed the basal diet for 2 or 4 weeks after the cessation of AAF-feeding.

Effects of NKK-105 against lipid peroxidation of microsomes by CCl4 are investigated, in vitro. NKK-105 had no effect on microsomal lipid peroxidation. When the microsomal fraction was preincubated with NKK-105 before adding CCl4, the lipid peroxidation induced with CCl4 was almost prevented by NKK-105. Effects of NKK-105 administration on aminopyrine N-demethylase, cyt.b5, cyt.p-45O and lipid peroxidation in CCl4-induced liver damage were investigated, in vivo. NKK-105 had no effect on the activity of aminopyrine N-demethylase and the content of cyt.b5, cyt.p-450 and peroxide. The content of cyt.b5, cyt.p-450 and the activity of demethylase remarkably decreased with CCl4 administration, but the content of cyt. p-450 and the demethylase activity were restored about 75% of the control values and cyt.b5 content was completely restored with previous administration of NKK-105. The amount of peroxide in the microsomal fraction increased with CCl4 administration was restored to the control level with previous administration of NKK-105.

During the early stages of the carcinogen 2-acetylaminofluorene (2-AAF) feeding, there were marked decreases of hepatic mitochondrial type B monoamine oxidase (MAO) and kynurenine 3-hydroxylase activities in male rats, but not in female rats. The administration of N-hydroxy-2-AAF (N-OH-2-AAF). a proximate carcinogenic metabolite of 2-AAF. to male rats also resulted in the decrease of both enzyme activities. The findings indicate that type B MAO and kynurenine 3-hydroxylase which reside in the outer mitochondrial membranes are susceptible to 2-AAF during the early stages of its hepatocarcinogenesis.

A rapid and sensitive method for determination of methamphetamine and amphetamine in urine was developed by using electron-capture gas chromatography. The extraction procedure, the experimental conditions for pentafluorobenzoyl derivative formation and the percentage recovery of the drugs from urine are described. The pentafluorobenzoyl derivative of methamphetamine showed a higher electron-capture sensitivity and was detected in at least 23-fold lower concentration than the heptafluorobutyryl derivative which is commonly used as a derivatizing agent for the amine. The detection limit of pentafluorobenzoyl derivatives of methamphetamine and amphetamine was ca. 10 pg. A concentration as low as 10 ng/ml of methamphetamine and amphetamine in urine was easily detected by this method.

A sensitive and specific gas chromatography/mass spectrometry (GC/MS) method has been developed for the simultaneous analysis of methampthetamine and its metabolites (amphetamine, p-hydroxymethamphetamine, and p-hydroxyamphetamine) in monkey urine. The method has been applied to the analysis of urine samples from monkeys singly and repeatedly treated with methamphetamine. We have shown that the repeated administration of methamphetamine causes the reduction of urinary excretion of aromatic hydroxylated metabolites. We propose that the method described here can be used for biological monitoring.

The effect of Pluronic F-127 (PF-127), a surfactant polyol, on the loading of fura 2/AM into smooth muscle cells isolated from guinea pig taenia coli was investigated. The presence of PF-127 during the loading of fura 2 acetoxymethyl ester (fura 2/AM), an intracellular Ca indicator, occasionally increased the incorporation of the dye into the cells. However, long time loading (over approximately 60 min) in the presence of PF-127 decreased the incorporation of fura 2. When the extracellular medium obtained from cells treated with DFP, then incubated with fura 2/AM in the presence of PF-127 was analyzed with excitation spectra, the fluorescence peaks shifted to longer wave lengths by addition of EGTA. However, the peak of the extracellular medium obtained from cells treated with DFP, then incubated without PF-127, did not shift. These results show that PF-127 affects the membranous permeability of the dye in single smooth muscle cells, permitting fura 2 hydrolyzed in the cytoplasm to leak out through the membrane.

The effects of a single and repeated administration of methamphetamine (MP) in vivo in rats on its own metabolism in vitro were investigated. In both cases, the p-hydroxylation of MP to p-hydroxymethamphetamine by a microsomal fraction from rat liver was inhibited for a period of 16 hr after the last injection of MP. This inhibition was diminished by dialysis of the microsomal preparations. In contrast, the reduced level of cytochrome P-450 in hepatic microsomes from rats pretreated with the SKF 525-A did not revert to the control value after dialysis. When microsomes were preincubated with N-hydroxymethamphetamine, which is the metabolite of MP and a potent substrate for the formation of a metabolic intermediate (MI) complex with cytochrome P-450, the content of the MI was increased and the MP-hydroxylation activity decreased in direct proportion to the length of the preincubation. These results suggest that the inhibition of MP-hydroxylation may be due to reduction of the level of cytochrome P-450 that accompanies the formation of the MI complex. Furthermore, it appears that the complex can be dissociated by dialysis.

The multiple forms of monoamine oxidase (MAO) in monkey brain were investigated using an electrofocusing technique. When beta-phenylethylamine (beta-PEA) was used as substrate, two peaks (peak I and peak II) could be clearly distinguished from the profile; the isoelectric point (pI) values were near 7.8 and 6.3, respectively. When serotonin (5-HT) was used, MAO activity was observed in peak I enzyme. The peak I enzyme with a pI value of 7.8 contains AB-form MAO and oxidizes 5-HT and beta-PEA, while peak II enzyme with a pI value of 6.3 contains B-form MAO and oxidizes beta-PEA, respectively. However, when peak II enzyme was incubated in a pH 8.8 reaction medium, MAO activity toward 5-HT in peak II enzyme was seen.

A rapid and sensitive gas-liquid chromatographic procedure was developed for the measurements of trimethadione (TMO) and its metabolite, 5, 5-dimethyl-2, 4-oxazolidine-dione (DMO) in plasma. TMO and DMO were extracted from plasma by a micro-extraction method and chromatographed on a 10% Carbowax 6000 column using paramethadione as an internal standard. Recoveries of TMO and DMO ranged from 96 to 104% using a 0.1 ml of plasma. The method is capable of measuring at least 5 microgram of TMO and DMO per milliliter. After an oral administration of TMO at a dose of 100 mg/kg, mean plasma levels of TMO and DMO in 6 rats reached a peak of 98 microgram/ml at 0.6 h and 163 microgram/ml at 8 h and declined with a half-life of 2.2 h and 39.4 h, respectively. After the intravenous administration of TMO at a dose of 100 microgram/kg, half-lives of TMO and DMO were 2.5 h and 39.1 h, respectively. Pretreatment of rats with carbon tetrachloride, d-galactosamine amd alpha-naphthyl isothiocyanate, prolonged T 1/2 and Tmax of TMO, reduced the Km value and increased the AUC. These results suggest that plasma levels of TMO and DMO might be useful as an index of drug metabolizing capacity in animal and man.

Methamphetamine N-demethylation and p-hydroxylation activities of rat liver were located mainly in the microsomal fraction. The Km values for methamphetamine and p-hydroxymethamphetamine demethylations were 1.0 and 1.6 mM, respectively. The Km value for amphetamine p-hydroxylation was 10.2 microM; substrate inhibition occurred at high substrate concn. Two Km values were obtained for the aromatic hydroxylation of methamphetamine (10.6 microM and 2.2 mM). N-Demethylation of methamphetamine and p-hydroxymethamphetamine were depressed in rats pretreated with 3-methylcholanthrene, CoCl2 or SKF 525-A. In rats pretreated with phenobarbital, methamphetamine demethylase was induced and p-hydroxymethamphetamine demethylase was depressed. The p-hydroxylation of methamphetamine and amphetamine in rats pretreated with phenobarbital, CoCl2, SKF 525-A or iprindole were depressed.

gamma-Hexachlorocyclohexane (gamma-HCH) was metabolized by dehydrochlorination, dehydrogenation and dechlorination in rat liver microsomes and these initial metabolites of gamma-HCH were identified as gamma-pentachlorocyclohexene (gamma-PCCH), gamma-hexachlorocyclohexene (gamma-HCCH) and gamma-tetrachlorocyclohexene (gamma-TCCH) by gas chromatography-mass spectrometry (GC/MS). The dehydrochlorination and dehydrogenation were performed in incubation media containing NADPH and p,p'-tetramethyldiaminodiphenyl methane (TPD, 30 nmol) which is known to inhibit the degradation of initial metabolites formed during an aerobic incubation of gamma-HCH with microsomes at 25 degrees C. The dechlorination was found to proceed well under anaerobic conditions. The dehydrogenation was inhibited by SKF 525-A, CO, piperonyl butoxide, N2 and the absence of NADPH, but not by cyanide. Additionally, pretreatment of rats with phenobarbital (PB), but not with 3-methylcholanthrene (3-MC), induced the dehydrogenation of gamma-HCH. These results suggest that cytochrome P-450 is involved in this reaction. The cytochrome b5 system may not be involved. The dehydrochlorination was inhibited by N2, CO, piperonyl butoxide, KCN and the absence of NADPH, but not by SKF 525-A. This reaction was enhanced by pretreatment of rats with SKF 525-A, CoCl2 and piperonyl butoxide. Pretreatment with PB and 3-MC did not show a significant effect on the dehydrochlorination activity. Thus, the results suggest that the dehydrochlorination could be catalyzed by a specific species of cytochrome P-450 and cytochrome b5 system and/or other microsomal enzyme systems.

After mice were given methamphetamine (MA) at a dose of 25 mg/kg i.p. twice daily (9:00 a.m. and 5:00 p.m.) for 4 days, the behavioral effects and releases of brain catecholamines induced by MA were investigated. The behavioral effects which were estimated according to the rating system of Peachey et al. were a predominant increase in locomotor activity at 2.5 mg/kg MA, a predominant stereotyped behavior at 10 mg/kg MA and the mixed type at 5 mg/kg MA in the saline control. Following repeated administration of MA, the rating scores of each effect at 2.5 and 5 mg/kg MA were not significantly different from those of the saline control. At 10 mg/kg MA, the behavioral effects were of the mixed type. However, when locomotor activity was measured by Automex II 2SD, significant increases in locomotor activity were observed over 2 hr. Following repeated administration of MA, the brain NE level did not alter, while the brain DA level was significantly decreased. The increased accumulation of brain MHPG-S0i+ induced by MA was not significantly different from the saline control. The accumulation of HVA, however, did not alter at high doses of MA, which caused the increased accumulation of HVA in the saline control.