Fatalities caused by the MDMA-related drug paramethoxyamphetamine (PMA).

The past several years have seen a marked increase in the recreational use of 3,4-methylenedioxymethamphetamine (MDMA) or "Ecstasy". MDMA use is especially common among young people participating in dance parties called "raves". Paramethoxyamphetamine (PMA) exhibits both structural and pharmacological similarity to MDMA. It may, however, be a more potent central stimulant, particularly in its effects on serotonergic transmission. Several fatalities from PMA have been reported in Australia, and here we report three recent fatalities that occurred in the midwestern United States in which each of the decedents believed that they were ingesting MDMA. Symptoms observed included agitation and bruxism, progressing to severe hyperthermia, convulsions, and hemorrhage. Blood was screened for drugs of abuse by enzyme immunoassay with the presence of amphetamines indicated in each case. Confirmation and quantitation for amphetamines was performed by gas chromatography-mass spectrometry. The deceased, two males ages 19 and 24 and a female age 18, had postmortem blood PMA concentrations of 1.07, 0.60, and 1.90 mg/L, respectively. PMA is not a contaminant of MDMA, and no MDMA was found in any of these cases. The primary metabolite of PMA is produced by O-demethylation to 4-hydroxyamphetamine, a reaction catalyzed by cytochrome P450 2D6. This enzyme is noted to be genetically polymorphic. Those with the "slow metabolizer" phenotype may be likely to have higher peak blood concentrations of PMA. Whether any of the decedents described herein were of the slow metabolizer phenotype is not known. Several groups have advocated the onsite use of the Marquis Test for the purpose of pill screening in efforts to distinguish PMA from MDMA. A dark purple is consistent with MDMA, whereas PMA imparts no color change in this test. PMA is often in the form of a white pill with a Mitsubishi symbol on one side. This design has been identified in at least one of these fatalities.

Induction of cytochrome P-450 enzymes after repeated exposure to 4-vinylcyclohexene in B6C3F1 mice.

4-Vinylcyclohexene (VCH), an ovarian toxicant in mice, is known to irreversibly deplete ovarian follicles as a consequence of VCH diepoxide formation. Because ovotoxicity requires repeated dosing of VCH, the effect of consecutive daily doses of VCH (7.5 mmol/kg/day) on mouse liver microsomal activities and VCH epoxidation was determined. Cytochromes P-450 2B and 2A (CYP2B and CYP2A), principle isoforms involved in the bioactivation of VCH, as well as CYP2E1 and CYP3A were evaluated. VCH exposure increased total cytochrome P-450 content (35-83% above control levels) after either 5, 10, or 15 days of treatment. Western blot analysis revealed an induction of CYP2A, CYP2B, and CYP2E1 at day 10. Elevated levels of CYP2A and CYP2B correlated with marker androstenedione and testosterone 16alpha- and 16beta-hydroxylase activities. Microsomes prepared from mice pretreated with VCH for 10 days demonstrated an increase (>/=2-fold) in the rate of VCH monoepoxide and diepoxide formation. Microsomal VCH epoxidation was increased to a similar extent by phenobarbital, acetone, and dexamethasone treatment. An increase in cytosolic glutathione S-transferase activity was observed after repeated VCH treatment, an enzyme potentially involved in detoxification of the VCH epoxides. Interestingly, preliminary studies indicated that circulating levels of the monoepoxide (vinylcyclohexene 1, 2-monoepoxide) and diepoxide of VCH were elevated after repeated dosing of VCH. Overall, the results indicate that repeated exposure of VCH in mice induces cytochrome P-450-dependent activities, and in turn induction of its metabolism. Additional studies examining the toxicokinetics of VCH after repeated exposure are required to further delineate the relevance of induction in VCH-induced ovotoxicity.

Suppression of rat hepatic microsomal cytochromes P450 by cyclophosphamide is correlated with plasma thyroid hormone levels and displays differential strain sensitivity.

Strain differences in cytochrome P450 (P450) expression were investigated in Sprague-Dawley (SDs) compared with Fischer 344s (F344s) rats after administration of cyclophosphamide (CPA). Animals received a single dose of CPA with sacrifice occurring 6 days post-treatment. At 130 mg/kg, male F344s displayed a greater sensitivity to CPA, as evidenced by a 68% loss of total hepatic microsomal P450 compared with only 35% in SDs. The most dramatic change in P450 was the loss of 2C11 (84% in F344s, 52% in SDs). In the SD, individual rat 2C11 activity was correlated (r2 = 0.76), with the level of plasma thyroxine in that animal. In male F344s administered CPA at 50 mg/kg, 43 and 44% losses in 2C11 activity (P < .05) and thyroxine (P < .01), respectively, were observed, whereas activities characteristic of P450s 2C11, 3A2, 2A2, 2C6 and 2E1/1A2 were unaffected in SDs at this dose. CPA also produced suppression of P450 in female SDs, including female-specific 2C12. Correlation was observed between the loss of P450 expression and change in body weight after treatment in both male and female animals, suggesting that CPA downregulates P450 expression secondary to decreased caloric intake. The anorectic effect of CPA is believed to result from potent central nervous system stimulation, accompanied by a state of adaptive hypothyroidism. It has been reported that CPA produces "feminization" of P450 expression in male rats. However, our findings suggest the alternative explanation that the effects of CPA on P450 expression result from decreased caloric intake.

Selective suppression of rat hepatic cytochrome P450 2C11 by chloramphenicol.

Chloramphenicol produces mechanism-based inactivation of several rat hepatic microsomal P450 enzymes including 2C6, 2C11, 2B1/2 and 3A1/2. A preliminary study by this laboratory reported that 48 hr after in vivo treatment with chloramphenicol (CAP) 2C11 activity remained low, whereas activities catalyzed by 3A2 and 2C6 were almost fully restored (Halpert et al., Biochem. Pharmacol. 37: 3046-3048, 1988). Therefore, in experiments conducted to examine whether CAP affects P450 expression, Sprague-Dawley (SD) rats were treated with CAP (single i.p. injection, 300 mg/kg) and sacrificed at various times post-treatment. The loss of P450 2C6, 2C11 and 3A2 catalytic activities which is characteristic of inactivation was demonstrated 1 hr after CAP administration. However, at 4 and 6 days, 2C11-mediated progesterone 2 alpha-hydroxylase activity remained diminished by 52 and 45%, respectively. Similar decreases in anti-P450 2C11-reactive protein and 2C11 mRNA were observed at 6 days, suggesting that the compound acts at a pretranslational step. Evaluation of 2C11 regulators indicated that CAP causes a decrease in plasma thyroxine level in proportion to the loss of 2C11 activity, whereas testosterone appears to be unaffected. To minimize intragroup variability, the inbred Fischer 344 strain was then examined at 2 and 6 days after CAP treatment. Surprisingly, CAP caused no loss in 2C11 protein, although the compound does inactivate 2C11 in liver microsomes from Fischer 344 rats. These results suggest that CAP alters P450 expression in a manner distinct from previously described compounds.

Induction of P4502E1 by acetone in isolated rabbit hepatocytes. Role of increased protein and mRNA synthesis.

The molecular mechanism(s) underlying induction of the hepatic microsomal cytochrome P4502E1 (2E1) by xenobiotics (e.g. ethanol and acetone) is controversial. Proposed mechanisms include increased rates of enzyme synthesis due to elevated 2E1 mRNA levels, enhanced translation of pre-existing mRNA, or stabilization of 2E1 protein. To further assess which, if any, of these events predominates during the initial stages of 2E1 protein induction, we investigated the effects of acetone treatment on 2E1 content in cultured rabbit hepatocytes, an in vitro system that allows for precise control of the cellular mileau. Hepatocytes harvested from female rabbits and plated on plastic dishes with serum-supplemented medium were 90-100% viable for at least 48 hr in culture. Analysis of immunoreactive 2E1 content and aniline hydroxylase activity in microsomes isolated from hepatocytes cultured for up to 24 hr revealed that 2E1 expression was equal to that of microsomes from unplated cells and by 48 hr of culture, 2E1 levels decreased by only 35%. Moreover, microsomes isolated from cells exposed to 17 mM acetone for 24 hr exhibited a 53 and 62% increase in aniline hydroxylase activity and 2E1 content, respectively, compared to untreated cells. To explain these increases, the rate of 2E1 protein synthesis was determined in untreated cells or in cells treated with 17 mM acetone by first exposing hepatocytes to medium supplemented with 35S-labeled methionine and cysteine ([35S]Met/Cys) and subsequently assessing radiolabel incorporation into 2E1 protein. While no difference was found between untreated and acetone-treated cells in the incorporation of [35S]Met/Cys into trichloracetic acid-precipitable microsomal proteins, immunoaffinity purification of 2E1 revealed that incorporation of 35S-labeled amino acids specifically into 2E1 was elevated by acetone to 200% of control values. Treatment of hepatocytes with the transcriptional inhibitor, alpha-amanitin, markedly inhibited this acetone-mediated increase in [35S]Met/Cys incorporation into 2E1. Analysis of hepatocyte RNA revealed that acetone increased 2E1 mRNA to 130 and 160% of control levels at 6 and 24 hr, respectively, and that these increases were prevented by pretreatment with alpha-amanitin. Our results indicate that acetone increases 2E1 protein levels in cultured rabbit hepatocytes by stimulating its rate of de novo synthesis. Since this increase in 2E1 synthesis stems, at least in part, from the acetone-mediated enhancement of hepatocyte 2E1 mRNA content and is inhibitable by alpha-amanitin, transcriptional activation of the rabbit CYP2E1 gene is apparently involved in the induction of 2E1 protein by acetone.

Bioactivation of halogenated hydrocarbons by cytochrome P4502E1.

Numerous halogenated hydrocarbons of the alkane, alkene, and alkyne classes are metabolized by P450 enzymes to products that elicit cytotoxic and/or carcinogenic effects. Such halogenated hydrocarbons include anesthetics (e.g., halothane and enflurane) and industrial solvents (e.g., carbon tetrachloride, chloroform, and vinylidine chloride). Formation of reaction intermediates from these compounds occurs via P450-promoted dehalogenation, reduction, or reductive oxygenation, with certain hydrocarbons undergoing all three reaction types. Of the multiple forms of P450 present in liver microsomes, P4502E1 has been identified as the primary catalyst of hydrocarbon bioactivation in animals and, most likely, in humans as well. As hepatic concentrations of this P450 enzyme are highly inducible by ethanol and similar agents, prior exposure to 2E1-inducing compounds can play a pivotal role in halogenated hydrocarbon toxicity. Considering that metabolism governs the cytotoxicity and carcinogenicity of halogenated hydrocarbons, an understanding of the mechanism(s) underlying 2E1 induction in man becomes all the more important.

Supplemental growth hormone alters body composition, muscle protein metabolism and serum lipids in fit adults: characterization of dose-dependent and response-recovery effects.

Using double-blind, placebo-controlled procedures, the effects of low and high therapeutic dosages of methionyl-human growth hormone (met-hGH) on body composition, muscle protein metabolism and serum lipids were studied in 7 fit adults without growth hormone (GH) deficiency. Dose-dependent changes in body composition were observed that in part appeared to be influenced by a response-recovery effect, as measured by responses factored according to the duration of washout between exposure to the low and high dosages of met-hGH (6 weeks vs. 12 weeks vs. 18 weeks). Increases in fat-free weight were accompanied by an increase in skeletal muscle protein metabolism. Basal levels of cholesterol were inversely related to peak levels of GH in response to exercise stimulation and IGF-I, while GH supplementation lowered levels of total cholesterol and high- and low-density lipoproteins. A dose-dependent effect occurred for total cholesterol, and the percent change in cholesterol was related to the percent change in insulin-like growth factor I (IGF-I). Endogenous levels of GH were attenuated in response to stimulation and IGF-I levels were increased after treatment with GH, but no dose-dependent changes were observed. We conclude that met-hGH alters body composition and muscle protein metabolism, and decreases stored and circulating lipids in fit adults with a pre-existing supranormal body composition. The physiological profile of the person was not as important as the treatment conditions in determining the somatic and physiological response outcomes.

Induction of cytochrome P450IIE1 in the obese overfed rat.

Cytochrome P450IIE1 (IIE1) is a microsomal xenobiotic-activating enzyme that is inducible not only by various chemical agents but also by fasting and diabetes. Using a rat model that mimics human obesity, we have found that hepatic IIE1 levels are also increased by this common clinical disorder. Liver microsomes from rats made obese by feeding with an energy-dense diet displayed elevated aggregate P450 content (+28%) and enhanced catalytic activities associated with IIE1, including low-Km N-nitrosodimethylamine demethylation (+66%), aniline hydroxylation (+52%), p-nitrophenol hydroxylation (+170%), and acetaminophen-cysteine conjugate formation (+28%). In contrast, obesity had no significant effect on cytochrome b5 content, P450 reductase activity, benzphetamine demethylation, or erythromycin demethylation, with the latter two reactions being linked with rat IIC11 and IIIA1, respectively. The enhancement of IIE1-dependent drug-metabolizing activities noted in liver microsomes from obese rats was paralleled by a similar increase (111%) in hepatic IIE1 protein content in these animals, as assessed on immunoblots developed with anti-hamster IIE1 IgG. Anti-IIE1-inhibitable rates of microsomal p-nitrophenol metabolism, a reaction highly correlated with IIE1 content (r = 0.88, p less than 0.01), were over 3-fold higher in obese rats than in nonobese controls, providing additional evidence for the obesity-related increase of hepatic IIE1. The induction of IIE1 by the pathophysiological condition of obesity may provide a biochemical basis for the increased incidence of occult liver disease and certain cancers noted in obese individuals.

Supplemental growth hormone increases the tumor cytotoxic activity of natural killer cells in healthy adults with normal growth hormone secretion.

Using double-blind, placebo-controlled procedures, the effects of methionyl-human growth hormone (met-hGH) on the tumor cytotoxic activity of natural killer (NK) cells were studied in seven healthy adults using a repeated measures experiment. Subjects were assigned at random to either a placebo (bacteriostatic water) treatment condition or a met-hGH (16.0 mg/wk of Protropin) treatment condition, then crossed-over to the alternative treatment. Treatments were delivered on alternate days (3 d/wk) for 6 weeks. Without bias from the met-hGH treatment, there was no evidence for GH hyposecretion as measured by the peak circulating GH response to exercise stimulation (14.1 +/- 3.1 ng/mL) or insulin-like growth factor (IGF-I) levels (0.82 +/- 0.09 U/mL). When compared with placebo, met-hGH induced a significant overall increase in the percent specific lysis (%SL) of K562 tumor target cells (placebo 22.2 +/- 1.7 v met-hGH 28.5 +/- 2.1 %SL; P = .008). NK activity was increased within the first week of treatment and this level was maintained throughout the remaining period of supplementation. There was a trend (P = .057) for the met-hGH-induced percent change in NK activity (NK%) to be inversely related to placebo IGF-I levels (r = -.761), while there were significant positive correlations between NK% and the met-hGH-induced percent changes in IGF-I (r = .727; P = .035), the fat-free mass (FFM)/fat mass (FM) ratio derived by hydrodensitometry (r = .792; P = .012), and the endogenous GH response to exercise (r = .469; P = .034).(ABSTRACT TRUNCATED AT 250 WORDS)

Exogenous growth hormone treatment alters body composition and increases natural killer cell activity in women with impaired endogenous growth hormone secretion.

In order to assess the potential relationship between human growth hormone (GH) and body composition (BC) and natural immunity (NI), we measured the effects of exogenous GH on fat weight (FW), fat-free weight (FFW), and the cytotoxic activity of natural killer (NK) cells in women with impaired GH secretion. Mean peak serum concentrations of GH in response to L-dopa/arginine stimulation were 6.2 +/- 1.1 (SEM) ng/mL in 6 untreated subjects (US) and 5.4 +/- 1.5 ng/mL in 6 GH-treated subjects (TS). Moreover, the pretreatment circulating levels of IGF-I were low in both groups (US 684 +/- 121 mU/mL and TS 583 +/- 83 mU/mL), and they correlated with pretest levels of NK cell activity (r = .59, P less than .05) when both groups were combined. The TS were given 700 micrograms of human GH IM for an average of 14 days while the US were studied in parallel without GH treatment. As measured by hydrodensitometry or skinfold anthropometry, FW decreased (26.1 +/- 6.8 kg to 23.8 +/- 6.3 kg, P less than .05) and FFW increased (44.9 +/- 3.3 kg to 46.2 +/- 3.8 kg, P less than .05) in the TS. In the US, there were no significant (P less than .05) changes in either FW or FFW. Using a standard 51Cr release assay to measure the specific lytic (SL) activity of NK cells, mean SL activity increased from 24.4 +/- 7.0% to 44.1 +/- 8.9% (P less than .05) in the TS, whereas levels in the US were not altered significantly (P less than .05).(ABSTRACT TRUNCATED AT 250 WORDS)