Separate mechanisms for procarbazine spermatotoxicity and anticancer activity.

Procarbazine causes dose-dependent decreases in sperm count after a single i.p. injection in (C57BL/6 X DBA/2)F1 male mice. Two antioxidants, N-acetylcysteine and sodium ascorbate, administered with equimolar doses of procarbazine decreased the spermatotoxicity of procarbazine. At the highest doses of procarbazine (400 mg/kg) that caused a 56% decrease in sperm count, equimolar doses of N-acetylcysteine coadministered with procarbazine caused only a 17% decrease in sperm count, and equimolar doses of ascorbate coadministered with procarbazine caused only a 13% decrease in sperm count. Thus, protection against the spermatotoxic effects of procarbazine was demonstrated with either antioxidant. The effect of the antioxidants on the chemotherapeutic efficacy of procarbazine against murine L1210 leukemia was also assessed. Procarbazine at the highest dose (600 mg/kg) increased mean survival time of mice inoculated i.p. with 1 X 10(5) L1210 leukemia cells by 31%. Simultaneous administration of equimolar doses of either N-acetylcysteine or ascorbate given with procarbazine caused no change in the increased mean survival time of tumor-bearing mice. These results indicate a decrease in the toxicity of procarbazine when coadministered with antioxidants, via decreased spermatotoxicity without changing anticancer efficacy. The results also indicate that different mechanisms are involved in the spermatotoxicity and anticancer activity of procarbazine.

Methylazoxyprocarbazine, the active metabolite responsible for the anticancer activity of procarbazine against L1210 leukemia.

Procarbazine is a 1,2-disubstituted hydrazine derivative that is used to treat human leukemias. The anticancer activity of procarbazine results from bioactivation to reactive intermediates. It is first oxidized to azoprocarbazine and further N-oxidized to a mixture of methylazoxyprocarbazine and benzylazoxyprocarbazine isomers. In this study the azoxyprocarbazine isomers were synthesized and purified. The cytotoxic effect of the metabolites on the L1210 murine leukemia cell line were then evaluated in vitro by use of a colorimetric assay using 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide. The results of this study showed that the methylazoxyprocarbazine isomer was the most cytotoxic metabolite (IC50, 0.2 mM). The benzylazoxy isomer had an insignificant cytotoxic effect, and a mixture of the two isomers was intermediate in effectiveness. This assay, however, could not be used to determine the cytotoxicity of procarbazine since the drug itself (not the live cells) reduced the dye. A soft-agar clonogenic assay demonstrated that procarbazine was cytotoxic only at higher concentrations (IC50, 1.5 mM) than methylazoxyprocarbazine (IC50, 0.15 mM). The effect of procarbazine and its metabolites on the survival of L1210 tumor-bearing mice was determined, and methylazoxyprocarbazine was again the most effective compound. These studies demonstrate that the methylazoxyprocarbazine metabolite is probably the major cytotoxic intermediate involved in the mechanism of anticancer action of procarbazine.

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.

Non-enzymatic activation of procarbazine to active cytotoxic species.

The cellular cytotoxicity of procarbazine is thought to result from bioactivation of the parent compound through reactive intermediates to an ultimate alkylating species. Procarbazine is converted initially to azoprocarbazine, which is then N-oxidized through a cytochrome P-450-mediated process to a mixture of the positional isomers, benzylazoxyprocarbazine and methylazoxyprocarbazine. In order to define the bioactivation events that lead to the cytotoxic species, the in vitro cytotoxicities of the purified azoxy isomers as well as of the parent compound, procarbazine, were evaluated with the human leukemia cell line, CCRF-CEM. The methylazoxy isomer was found to be the most active species. Procarbazine inhibited the growth of CCRF-CEM cells but at a concentration much higher than that required for the methylazoxy isomer. Since procarbazine must be metabolized to form the cytotoxic species, we sought to determine if the active metabolite, methylazoxyprocarbazine, was being formed in the incubations. Solutions of procarbazine incubated with and without cells at 37 degrees C were analyzed by combined liquid chromatography-mass spectrometry with a thermospray interface. The azoxy metabolites of procarbazine appeared rapidly in cellular incubations and in the aqueous solutions without cells. More of the methylazoxy isomer was formed initially, but by 72 hr the benzylazoxy isomer was the predominant species. Thus, in these studies it appears that procarbazine was benzylazoxy isomer was the predominant species. Thus, in these studies it appears that procarbazine was non-enzymatically oxidized to the two azoxyprocarbazine isomers and that the methylazoxy compound was the most cytotoxic to CCRF-CEM cells.

Determination of capsaicin, dihydrocapsaicin, and nonivamide in self-defense weapons by liquid chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry.

Sensitive and selective liquid chromatography-mass spectrometry (LC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS-MS) methods for the analysis of capsaicin, dihydrocapsaicin, and nonivamide in pepper spray products have been developed. Chromatographic separation of the capsaicinoid analogues was achieved using a reversed-phase HPLC column and a stepwise gradient of methanol and distilled water containing 0.1% (v/v) formic acid. Identification and quantification of the capsaicinoids was achieved by electrospray ionization single-stage mass spectrometry monitoring the protonated molecules of the internal standard (m/z 280), capsaicin (m/z 306), dihydrocapsaicin (m/z 308), and nonivamide (m/z 294) or by tandem mass spectrometry monitoring the appropriate precursor-to-product-ion transitions. The plot of concentration versus peak area ratio was linear over the range of 10-750 ng/ml using LC-MS and 10-500 ng/ml using LC-MS-MS. However, to accurately quantify the capsaicinoids in the pepper spray products calibration curves between 10 and 1000 ng were constructed and fit using a weighted quadratic equation. Using the quadratic curve, the accuracy of the assay ranged from 91 to 102% for all analytes. The intra-assay precision (RSD) for capsaicin was 2% at 25 ng/ml, 10% at 500 ng/ml, and 3% at 800 ng/ml. The inter-assay precision (RSD) for capsaicin was 6% at 25 ng/ml, 6% at 500 ng/ml, and 9% at 800 ng/ml. Similar values for inter- and intra-assay precision were experimentally obtained for both dihydrocapsaicin and nonivamide. The analysis of selected pepper spray products demonstrated that the capsaicinoid concentration in the products ranged from 0.7 to 40.5 microg/microl.

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.

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.

Correlation between pulmonary cytochrome P450 transcripts and the organ-selective pneumotoxicity of 3-methylindole.

3-Methylindole (3MI) is a species- and organ-selective pneumotoxin; goats are the most susceptible species, mice are intermediate in susceptibility, and rabbits are the least susceptible species to its toxicity. Four different cDNA probes representative of human cytochrome P450 genes CYP2F1, CYP4B1, CYP2A6, and CYP2B6 were hybridized against RNA from lung and liver tissues of goat, mouse and rabbit. Transcripts representative of pulmonary P450s CYP2F1, CYP4B1 and CYP2B6 were present in goat lung. Transcripts for the CYP2F1 and CYP4B1 genes were observed in rabbit and mouse lung. In general, the probes selectively hybridized to pulmonary but not hepatic transcripts of all three species. The differences in susceptibilities among the three species could not be explained by the lack of 4B1 and 2F1 transcripts in the lungs of mice or rabbits that are less susceptible than goats, but the selective expression in the lung tissues of all three species may participate in the organ-selective bioactivation and pulmonary toxicity of 3MI in these species.

Effect of melengestrol acetate on development of 3-methylindole-induced pulmonary edema and emphysema in sheep.

The involvement of melengestrol acetate (MGA) in susceptibility to developing pulmonary edema and emphysema following oral administration of 3-methylindole (3MI) was investigated using 10 Suffolk ewes receiving 0 or 0.15 mg of MGA daily (n = 5). Blood, urine and ruminal fluid were collected immediately prior to 3MI dosing (0.2 g/kg BW) and 1, 2, 3, 4, 5, 6, 12 and 24 h (blood); 3, 6, 9, 12 and 15 h (urine) and 1, 2, 3 and 12 h (ruminal fluid) afterward. Ewes receiving MGA experienced earlier (P < 0.05) onset of respiratory distress than the control ewes (2.5 vs 4 h), and upon euthanasia at 96 h, their lung weight relative to body weight tended (P < 0.10) to be lower. Ruminal 3MI concentrations did not differ between treatments (P > 0.05). Ewes receiving MGA had higher (P < 0.05) concentrations of 3MI metabolites in plasma prior to dosing than did control ewes, and these values tended to remain higher throughout the sampling period. Immunoreactivity assays indicated more pneumotoxin present in the lungs of MGA-treated ewes than controls. Lung damage was apparently more acute and accelerated in the MGA-treated ewes than in the controls. Urinary 3MI mercapturate concentrations differed (control > MGA-treated, P < 0.05) at 9, 12, and 15 h, but this difference was not apparent when urinary production (as estimated by creatinine concentration) was considered. The implications of these findings for MGA-treated feedlot heifers are currently under investigation.

Involvement of microbial respiratory pathogens in acute interstitial pneumonia in feedlot cattle.

OBJECTIVE: To evaluate viral and bacterial respiratory pathogens and Mycoplasma spp isolated from lung tissues of cattle with acute interstitial pneumonia (AIP) and cattle that had died as a result of other causes. SAMPLE POPULATION: 186 samples of lung tissues collected from cattle housed in 14 feedlots in the western United States. PROCEDURE: Lung tissues were collected during routine postmortem examination and submitted for histologic, microbiologic, and toxicologic examinations. Histologic diagnoses were categorized for AIP, bronchopneumonia (BP), control samples (no evidence of disease), and other disorders. RESULTS: Cattle affected with AIP had been in feedlots for a mean of 1272 days before death, which was longer than cattle with BP and control cattle. Detection of a viral respiratory pathogen (eg, bovine respiratory syncytial virus [BRSV], bovine viral diarrhea virus, bovine herpesvirus 1, or parainfluenza virus 3) was not associated with histologic category of lung tissues. Bovine respiratory syncytial virus was detected in 8.3% of AIP samples and 24.0% of control samples. Histologic category was associated with isolation of an aerobic bacterial agent and Mycoplasma spp. Cattle with BP were at greatest risk for isolation of an aerobic bacterial agent and Mycoplasma spp. CONCLUSIONS AND CLINICAL RELEVANCE: Analysis of these results suggests that AIP in feedlot cattle is not a consequence of infection with BRSV. The increased, risk of isolation of an aerobic bacterial agent from cattle with AIP, compared with control cattle, may indicate a causal role or an opportunistic infection that follows development of AIP.

Association of 3-methyleneindolenine, a toxic metabolite of 3-methylindole, with acute interstitial pneumonia in feedlot cattle.

OBJECTIVE: To compare concentrations of 3-methyleneindolenine (3MEIN) in lung tissues obtained from feedlot cattle that died as a result of acute interstitial pneumonia (AIP) and cattle that died as a result of other causes and to compare blood concentrations of 3MEIN in healthy feedlot cattle and feedlot cattle with AIP. STUDY POPULATION: Blood samples and lung tissues collected from 186 cattle housed in 14 feedlots in the western United States. PROCEDURE: Samples of lung tissues were collected during routine postmortem examination and submitted for histologic, microbiologic, and toxicologic examination. Blood samples were collected from cattle with clinical manifestations of AIP and healthy penmates. Histologic diagnoses were categorized as AIP, bronchopneumonia (BP), control samples, and other disorders. Concentrations of 3MEIN were determined in lung tissues and blood samples, using an ELISA. RESULTS: Concentrations of 3MEIN in lung tissues were significantly greater in AIP and BP samples, compared with control samples. Absorbance per microgram of protein did not differ between BP and AIP samples. Blood concentrations of 3MEIN were significantly greater in cattle with AIP, compared with healthy cattle or cattle with BP. Odds of an animal with AIP being a heifer was 3.1 times greater than the odds of that animal being a steer. CONCLUSIONS AND CLINICAL RELEVANCE: Increased pulmonary production of 3MEIN may be an important etiologic factor in feedlot-associated AIP.

Quantitative analysis of capsaicinoids in fresh peppers, oleoresin capsicum and pepper spray products.

Liquid chromatography-mass spectrometry was used to identify and quantify the predominant capsaicinoid analogues in extracts of fresh peppers, in oleoresin capsicum, and pepper sprays. The concentration of capsaicinoids in fresh peppers was variable. Variability was dependent upon the relative pungency of the pepper type and geographical origin of the pepper. Nonivamide was conclusively identified in the extracts of fresh peppers, despite numerous reports that nonivamide was not a natural product. In the oleoresin capsicum samples, the pungency was proportional to the total concentration of capsaicinoids and was related by a factor of approximately 15,000 Scoville Heat Units (SHU)/microg of total capsaicinoids. The principle analogues detected in oleoresin capsicum were capsaicin and dihydrocapsaicin and appeared to be the analogues primarily responsible for the pungency of the sample. The analysis of selected samples of commercially available pepper spray products also demonstrated variability in the capsaicinoid concentrations. Variability was observed among products obtained from different manufacturers as well as from different product lots from the same manufacturer. These data indicate that commercial pepper products are not standardized for capsaicinoid content even though they are classified by SHU. Variability in the capsaicinoid concentrations in oleoresin capsicum-based self-defense weapons could alter potency and ultimately jeopardize the safety and health of users and assailants.

Characterization of acute interstitial pneumonia in cattle in southern Alberta feedyards.

Field data were collected over 2 consecutive years to characterize acute interstitial pneumonia (AIP) in feedyard cattle. Thirty-eight cattle with clinical symptoms of AIP were examined following emergency slaughter; 31 (all heifers) were confirmed to have AIP on the basis of gross and histological lung pathology. The 7 without AIP, plus 17 asymptomatic penmates, were used as contemporary controls. Plasma concentrations of 3-methylindole (3MI) metabolites were higher (P < 0.001) in heifers afflicted with AIP than in the control animals, and concentrations of 3MI mercapturates in the urine were lower (P < 0.007) in affected heifers. Concentrations of 3MI adducts in lung tissue and in microsomal protein did not differ (P > 0.05) between the 2 groups, and 3MI was not detected in ruminal fluid from either group. Total ruminal bacterial numbers and populations of lactobacilli and protozoa were similar (P > 0.05) between the AIP-positive and unafflicted groups, but fewer (P < 0.05) cellulolytic bacteria were present in the positive group. Bovine respiratory syncytial virus antigen was not found in lung tissue from any of the heifers confirmed to have AIP. To our knowledge, this study is the first to implicate 3MI metabolites as having a role in feedyard AIP. Further research is required to determine the factors responsible for the elevation in 3MI adducts in plasma and urine of feedyard cattle afflicted with AIP.

Purification and characterization of an ethanol-induced UDP-glucuronosyltransferase.

A UDP-glucuronosyltransferase (GT) enzyme was isolated from ethanol-induced male New Zealand white rabbit hepatic protein. The animals were pretreated for 2 weeks with 10% ethanol in their drinking water. The GT enzyme was purified by anion-exchange and affinity chromatography and was shown to be homogeneous by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. The molecular mass of the ethanol-induced UDP-glucuronosyltransferase was determined to be 57,000 Da. Tryptic digests of the ethanol-induced GT and a similarly purified GT from control rabbit liver appeared to be different by HPLC analysis, even though the molecular masses of the enzymes were indistinguishable. Amino acid compositions of the two proteins were different for six amino acids. The apparent Km values for the ethanol-induced GT enzyme for 1-naphthol and morphine as substrates were 43 and 109 microM, respectively. The apparent Vmax values for the ethanol-induced GT enzyme for these substrates were 83 and 4.6 nmol/min/mg protein. The increases in catalytic efficiencies, apparent Vmax/Km for 1-naphthol and for morphine, for the ethanol-induced isozyme compared to the control isozyme activities were 2.0- and 2.4-fold. A polyclonal antibody raised in sheep to the rabbit ethanol-induced GT demonstrated a 520-fold selectivity for precipitation of the ethanol-induced protein rather than the control protein. These results demonstrate the production of an unique isozyme of UDP-glucuronosyltransferase that is produced in rabbits as a result of chronic ethanol exposure.

Production and characterization of specific antibodies: utilization to predict organ- and species-selective pneumotoxicity of 3-methylindole.

3-Methylindole (3MI) selectively causes damage to pulmonary tissues; the species-selective order is goats, rats, and rabbits, with rabbits sustaining the least damage. 3MI is bioactivated to toxic intermediates by cytochrome P450 enzymes. Covalent binding of the electrophilic 3-methyleneindolenine intermediate to proteins is a likely mechanism of 3MI-mediated lung damage. Polyclonal antibodies were developed to thioether adducts of 3-methyleneindolenine and were shown by competitive enzyme-linked immunosorbent assay (ELISA) to be highly selective for the detection of 3MI adducts. Rabbits, rats, and goats were treated with 350, 400, and 15 mg/kg 3MI, respectively. The lungs, liver, and kidneys of each animal were collected 24 hr later and tissue fractions were analyzed by ELISA. Lung tissue fractions from goat (pellet, cytosol, and microsomes) had greater immunoreactivity than those from rat. Immunoreactivity in rat tissues was greater than that in rabbit tissues. In all of the animals, lung had greater immunoreactivity than kidney, and kidney had greater reactivity than liver. These studies demonstrate that thioether adducts of 3MI with proteins can be detected specifically by these antisera, and the adducts are precisely correlated to species and tissue susceptibility of 3MI. In addition, human lung and liver samples were moderately immunoreactive. Therefore, humans form adducts of 3MI in these tissues and are predicted to be susceptible to 3MI-mediated toxicity.