Use of the vial equilibration technique for determination of metabolic rate constants for dichloromethane.

Metabolism of methylene chloride, or dichloromethane (DCM), plays a key role in determining the kinetics and carcinogenicity of the halocarbon. The objectives of this study were: to evaluate and optimize the vial equilibration technique, originally described by Sato and Nakajima (1979a), in order to characterize the hepatic metabolism of DCM by Sprague-Dawley rats; to employ different hepatic microsomal preparations to examine buffer effects on DCM metabolism; and to assess the relative importance and metabolic constants of the mixed-function oxidase (MFO) and glutathione (GSH) S-transferase (GST) metabolic pathways. A crude liver homogenate (20% W/V) was prepared from perfused livers of male Sprague-Dawley (S-D) rats (275-325 g). A 30% glycerol buffer was found to significantly inhibit DCM metabolism, while 0.25 M sucrose buffer containing 10 mM EDTA and 1.15% KCl did not. DCM was incubated with the liver 10,000 g supernatant or microsomes and cofactors in sealed headspace vials. Disappearance of DCM, as a measure of the chemical's metabolism, was monitored by headspace gas chromatography. Different trials were conducted to elucidate time-, enzyme-, and substrate-activity relationships. The scaled-up K(m) and Vmax values for the microsomal fraction were quite similar to optimized in vivo values reported by other investigators. In the current study, DCM appeared to be metabolized preferentially by cytochrome P450 IIE1, since substrates (e.g., pyrazole, ethanol, and glycerol) for this isozyme completely inhibited DCM metabolism. Thus, glycerol should not be used as a P450 stabilizer for preparation or storage of microsomes. Phorone pretreatment caused marked hepatic GSH depletion, but had little effect on the overall rate of DCM metabolism. Quantitatively, the GST pathway in the cytosol played a very minor role in DCM metabolism. It was not possible to accurately calculate metabolic constants for this pathway in S-D rats. The vial equilibration technique, as described here, is a relatively simple and reliable method, which should be broadly applicable for measuring the microsomal metabolism of DCM and other VOCs.

A comprehensive evaluation of the vial equilibration method for quantification of the metabolism of volatile organic chemicals. Trichloroethylene.

A study was undertaken, using 1,1,2-trichloroethylene (TCE), to: a) evaluate and improve the efficiency of the vial equilibration method, as first reported by Sato and Nakajima (Toxicol. Appl. Pharmacol. 47, 41-46, 1979); b) select an appropriate vehicle for the volatile substrate; c) examine the rate of metabolism of TCE by different rat hepatic microsomal preparations; d) determine TCE metabolic rate constants; and e) assess the influence of oleic acid and linoleic acid (the two major fatty acids in corn oil) on TCE metabolism. TCE was incorporated into a 1% Alkamuls aqueous emulsion and incubated with cofactors and the 10,000g supernatant or the microsomal fraction of livers of male Sprague-Dawley rats (275-325 g) in sealed headspace vials. Different trials were conducted to elucidate time-, enzyme-, and substrate-activity relationships. The apparent Km and Vmax were 2.0 microM and 1.9 nmol/mg protein/10 min for substrate concentrations ranging from 0.3 to 34 microM in the 10,000g fraction. The apparent Km and Vmax for the microsomal fraction were 4.17 microM and 8.0 nmol/mg protein/10 min, respectively. Linoleic acid concentrations of 10 microM and higher reduced TCE metabolism by the 10,000g fraction, whereas oleic acid concentrations of 50 microM and higher were required to inhibit TCE metabolism. Isooctane and dimethylsulfoxide caused a significant decrease in TCE metabolism. The vial equilibrium technique, as described herein, is relatively simple, rapid, and reliable, and may be applicable as a standard procedure for quantifying volatile organic chemical metabolism by different tissues of different species, including humans.

Distribution of cardiac output during diurnal changes of activity in rats.

Rat locomotor and feeding behavior varies on a diurnal basis; at night the animals actively forage and eat, whereas during the day they are more inactive and somnolent. At night, cardiac output is higher, presumably for enhanced perfusion of the active muscles to support increased metabolism and for enhanced perfusion of the digestive organs to support increased digestion and nutrient absorption. Conversely, it is hypothesized that during the daytime, blood flow to these two tissues is relatively low. The purpose of this study was to test these hypotheses by measuring cardiac output and the distribution of cardiac output in rats at various times in the diurnal cycle (8:00 A.M., 4:00 P.M., and 8:00 P.M.). The radiolabeled microsphere technique was used to measure cardiac output and distribution of blood flow to the tissues. Distribution of the total cardiac output was accounted for by complete dissection, weighing, and counting of organs and carcass. Cardiac output at 8:00 P.M. (136 +/- 9 ml/min) was elevated 13% (P less than 0.05) over that at 4:00 P.M. The proportion of the cardiac output distributed to the skeletal muscles (4:00 P.M.: 25%; 8:00 P.M.: 27%) and to the digestive tract (4:00 P.M.: 14%; 8:00 P.M.: 14%) did not change between the two time periods. Thus total muscle blood flow increased (P less than 0.05) from 31 +/- 2 at 4:00 P.M. to 36 +/- 4 ml/min at 8:00 P.M.; the only digestive organ to show a significant increase in blood flow from 4:00 P.M. to 8:00 P.M. was the stomach (133 +/- 17 to 166 +/- 19 ml/min, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolism and disposition of sulfluramid, a unique polyfluorinated insecticide, in the rat.

The objectives of this study were to characterize the absorption, distribution, and elimination of sulfluramid (N-ethyl perfluorooctane sulfonamide) and its major metabolite, perfluorooctane sulfonamide (DESFA), in order to assess the effect of dosage vehicle on their pharmacokinetics. In Trial 1, male and female Sprague-Dawley rats (170-240 g) were housed in Roth-type metabolism cages. Each rat received 50 mg/kg sulfluramid po, which contained 10 microCi of [14C] sulfluramid. Feces, urine, and expired air samples were collected for 72 hr post-dosing. Tissue samples also were collected at 72 hr and 14C distribution determined. Eighty percent of the radiolabel was eliminated within 72 hr, with the largest quantities of 14C recovered in expired air (56%) and feces (25%). Less radiolabel was recovered in urine (8%), and even smaller amounts in tissues (5%). The highest tissue concentrations of 14C were found in liver, kidneys, and adrenals, with significantly more radiolabel in the kidneys, gonads, and adrenals of the females than males. Male Sprague-Dawley rats (250-275 g) were used in Trials 2 and 3. In Trial 2, rats with a carotid artery cannula were given 50 mg/kg sulfluramid in a po bolus of polyethylene glycol 400 (PEG) or corn oil and blood samples were collected for 96 hr. In Trial 3, noncannulated rats were given 50 mg/kg sulfluramid po in PEG or corn oil and blood samples were collected from the caudal artery for 14 days. Blood samples were analyzed for sulfluramid and DESFA by gas chromatography.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of phenobarbital and beta-naphthoflavone on the in vivo toxicity and in vitro metabolism of aflatoxin in an aflatoxin-resistant and control line of chickens.

The in vivo toxicity of aflatoxin and the in vitro microsomal metabolism of aflatoxin B1 (AFB1) were investigated in a population of chickens previously selected for resistance to aflatoxin (AR line) and a corresponding control population (NS line) after in vivo pretreatment with saline, sodium phenobarbital (PB), or beta-naphthoflavone (BNF) solutions. PB pretreatment increased survival and BNF pretreatment increased mortality in both the NS and AR lines when a single oral dose of aflatoxin was administered. The rate of in vitro metabolism of AFB1 was greater with microsomes from saline pretreated AR chicks than with microsomes from similarly treated NS chicks. In vivo pretreatment with PB increased AFB1 metabolism by NS and AR microsomes. After BNF pretreatment of vivo, AR microsomes metabolized more AFB1 than NS microsomes, and there was a dramatic decrease in AFB1 metabolism in NS microsomes. AFB1-dihydrodiol was the major metabolite produced by both lines, with aflatoxin M1 and aflatoxin Q1 recovered in small quantities from BNF-pretreated AR microsomal incubations only. These data indicate that increased in vivo resistance of the AR line to acute aflatoxicosis may be related to increased hepatic AFB1 metabolism and that genetic selection has resulted in altered in vitro quantitative and qualitative metabolism of AFB1 in the AR line.

Effects of dietary aflatoxin in aflatoxin-resistant and control lines of chickens.

The resistance to a single oral dose (12 mg of aflatoxin per kg of BW) and 4 wk of dietary aflatoxin (2.5 mg per kg of feed) were investigated in chicks selected for five generations for resistance to acute aflatoxicosis (AR) and unselected control (C) chicks. The AR chicks were more resistant to a single oral dose of aflatoxin and had significantly decreased sodium pentobarbital sleeping time compared to C chicks. Four-weeks exposure to dietary aflatoxin did not result in any significant change in BW or feed conversion ratios of chicks from either the C or AR line. However, more sensitive indicators of aflatoxicosis including plasma total protein, albumin, cholesterol concentrations, and gamma glutamyl transferase activity were significantly altered in C chicks but not in AR chicks fed aflatoxin. Percentages of liver lipid and liver hyperplasia score were also significantly altered as a result of dietary aflatoxin treatment in C but not AR chicks. These data indicate that selection-associated differences exist between the C and AR lines of chickens that convey resistance to not only a single oral dose of aflatoxin but also to a more chronic dietary exposure to aflatoxin.

Effect of cold acclimation on the broiler chicks' resistance to dietary aflatoxin.

The effect of acclimation to environmental temperatures of 10 to 12 or 28 to 30 C on the resistance of broiler chicks to dietary aflatoxin was examined. Broiler chicks were acclimated from day-of-age for 2 wk to environmental temperatures of 10 to 12 or 28 to 30 C. On Day 14, a single oral dose of aflatoxin (8 mg per kg of body weight) was administered to 50 chicks in each environment. An increase in aflatoxin resistance, as assessed by survival rate, was conveyed by acclimation to cold temperatures. In each environmental chamber, a separate group of chicks was maintained for 2 additional wk, but those groups received 5 mg of aflatoxin per kg feed. By the end of the study, aflatoxicosis was characterized by: 1) a significant (P less than or equal to .05) decrease in body weight; 2) increases in spleen weight, liver weight, liver lipid, and liver dry-matter content; 3) changes in the serum levels of total protein, albumin, glucose, cholesterol, uric acid, potassium, phosphorus, iron and calcium; and 4) increased hepatic hyperplasia. Acclimation to 10 to 12 C was characterized by: 1) an increase in body weight, liver weight, spleen weight and bursa weight; 2) changes in the serum glucose and potassium levels; and 3) a decrease in glutamic-oxaloacetic transaminase activity. Significant aflatoxin by temperature interactions were evident only in serum levels of glucose and phosphorus, and in the serum activity of glutamic-oxaloacetic transaminase. These data suggest that acclimation to cool temperatures does not play a significant role in the resistance by broiler chickens to chronic aflatoxin exposure.

Perfluorooctane sulfonamide: a structurally novel uncoupler of oxidative phosphorylation.

The effects of sulfluramide (N-ethylperfluorooctane sulfonamide) and perfluorooctane sulfonamide (DESFA) on isolated rabbit renal cortical mitochondria (RCM) were examined. Sulfluramid (1-100 microM) and DESFA (0.5-50 microM) increased state 4 respiration of RCM respiring on pyruvate/malate or succinate in a concentration dependent manner in the absence of a phosphate acceptor. In addition, both sulfluramid and DESFA increased state 4 respiration in the presence of oligomycin, an inhibitor of F0F1-ATPase. The effects of sulfluramid (200 microM), DESFA (100 microM), and the known protonophore and uncoupler of oxidative phosphorylation, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) (1 microM), on RCM proton movement were examined directly by monitoring extramitochondrial pH and indirectly by monitoring passive mitochondrial swelling. Immediately upon addition, DESFA and FCCP, but not sulfluramid, dissipated the RCM proton gradient and caused RCM to swell in solutions of NaCl or NH4Cl. These results show that DESFA uncouples oxidative phosphorylation by acting as a protonophore. RCM were shown to metabolize sulfluramid to DESFA which suggests that the increase in state 4 respiration observed with sulfluramid is due to DESFA. DESFA is unique in that it is one of two uncouplers that does not contain a ring structure and thus may be a useful model in the study of oxidative phosphorylation.

Effect of cold acclimation on the broiler chicks' resistance to acute aflatoxicosis.

The effect of acclimation to environmental temperatures of 10 to 12, 14 to 16, or 28 to 30 C on the resistance of broiler chicks to acute aflatoxicosis was studied in a series of four experiments. Chicks acclimated to 10 to 12 C were more resistant to a single oral dose of aflatoxin (8 mg per kg of BW) after 10 and 20 days than chicks acclimated to 28 to 30 C. However, chicks acclimated to 14 to 16 C required 20 days of acclimation before an increase in resistance was observed. Acclimation to 10 to 12 C for 14 days followed by acclimation to 28 to 30 C for 7 days resulted in no increase in the resistance of the chicks to an aflatoxin dose (8 mg per kg of BW) at 21 days, indicating that the resistance conveyed by cold acclimation was temporary. Sodium-pentobarbital sleeping time was significantly decreased and the cytochrome P-450 content was significantly increased after acclimating broiler chicks to 10 to 12 C. These observations suggest that stimulation of the microsomal mono-oxygenase system after cold acclimation may be responsible for the increased resistance of the broiler chick to acute aflatoxicosis.

Selection for resistance to aflatoxin in chickens.

Two populations of broiler chickens [Athens-Canadian (AC) and a commercial stock] were selected for resistance to aflatoxicosis by dosing chickens in each population with a single oral dose of aflatoxin, which resulted in 40 to 70% mortality. Chickens that did not receive aflatoxin served as the nonselected control groups. Birds surviving the aflatoxin challenge were used as breeders for subsequent generations. Rapid progress was made in the AC population for resistance to aflatoxin, but only moderate progress for this trait was attained in the commercial broiler stock. After five generations of selection in the AC population, LD50 values of 9.42 and 17.05 milligrams aflatoxin per kilogram body weight (BW) were determined for the nonselected and selected lines, respectively. After four generations of selection in the commercial broiler population, LD50 values of 6.05 and 8.02 mg aflatoxin/kg BW were determined for the nonselected and selected lines, respectively. These data demonstrate that genetic progress can be made in chickens, but the amount of progress for resistance to aflatoxin may be influenced by the population.

The individual and combined effects of citrinin and ochratoxin A on renal ultrastructure in layer chicks.

Layer chicks fed 3.0 micrograms of ochratoxin A (OA) and 300 micrograms of citrinin (CTN) per gram of feed, alone and in combination, were evaluated for changes in renal ultrastructure. Feeding OA from 0 to 21 days of age was associated with proximal tubular intranuclear membrane-bound inclusions, elongated tortuous and ring-shaped mitochondria, enlarged mitochondrial matrix granules with hyaline centers, and an increase in number and size of peroxisomes and secondary lysosomes. Birds fed OA from 0 to 7 days and then given untreated ration had similar changes but to a lesser degree. Feeding CTN from 0 to 21 days of age was associated with similar proximal tubular nuclear inclusions, elongated tortuous and ring-shaped mitochondria, and an increase in size and number of peroxisomes and secondary lysosomes. Hyalinized mitochondrial matrix granules were not present, and some proximal tubular cells had cytoplasmic aggregates of smooth endoplasmic reticulum. Birds fed CTN from 0 to 7 days had similar but milder changes. Birds fed CTN + OA for 21 days had changes similar to those fed OA alone and also had cytoplasmic aggregates of smooth endoplasmic reticulum similar to those of CTN-fed birds. Again, changes in birds fed CTN + OA for 7 days were similar but milder.

The individual and combined effects of citrinin and ochratoxin A in broiler chicks.

Citrinin (CTN) and ochratoxin A (OA) were fed alone and in combination to broilers from day of hatch until 3 weeks of age. Dietary concentrations of 300 micrograms CTN/g and 3.0 micrograms OA/g were used. Birds fed CTN had significantly (P less than or equal to 0.05) lower body weights than controls on days 14 and 21 and increased water consumption on days 7, 14, and 21. Birds fed OA had significantly lower body weights than controls on days 7, 14, and 21 and increased water consumption on day 14. Birds fed CTN and OA in combination had lower body weights than controls and increased water consumption during the experiment, but the alterations were intermediate in severity when compared with those in birds fed CTN or OA alone. Birds fed OA alone or combined with CTN had higher liver and kidney weights than controls, but birds fed CTN alone had only higher kidney weights. Birds fed both CTN and OA had concentrations of serum constituents similar to those in birds fed OA alone, except the levels of cholesterol and triglycerides were not significantly different from those in the controls. Histological evaluation of the kidney indicated no lesions in birds fed CTN alone, but birds fed OA, alone or in combination with CTN, had increased tubular casts and tubular hyperplasia compared with controls. These data suggest that there were no additive or synergistic toxic interactions when 300 micrograms CTN/g and 3.0 micrograms OA/g were fed simultaneously to broiler chicks for 3 weeks. However, the severe growth depression resulting from OA and the increased water consumption associated with CTN toxicosis were ameliorated when CTN and OA were fed in combination. These data may be useful in diagnosing field cases of mycotoxicosis where both CTN and OA are involved.

Toxicity of Aspergillus ochraceus contaminated wheat and different chemical forms of ochratoxin A in broiler chicks.

The toxicity of Aspergillus ochraceus contaminated wheat and various chemical forms of ochratoxin A (OA) were compared by feeding diets containing A. ochraceus contaminated wheat (3.0 micrograms OA/g diet) and 3.0 micrograms/g of purified OA in the K salt, Na salt, or organic acid form to broiler chicks from hatching to 4 weeks of age. All OA diets caused listlessness, emaciation, dehydration, and occasional diarrhea. Cumulative mortalities were 0, 13, 17, and 10% for the control, contaminated wheat, OA Na salt, OA K salt, and OA acid, respectively. Necropsies at the end of the experiment revealed pale friable livers, enlarged pale kidneys, and enlarged gall bladders in all OA treatments. Body weights and gain were significantly depressed throughout the experiment, and cumulative feed conversion ratios were significantly increased by all forms of OA. Relative kidney and liver weights were also significantly increased by all forms of OA. Serum analysis revealed significant decreases in total protein, albumin, globulin, cholesterol, and phosphorus concentrations and significant increases in uric acid concentrations in chicks fed all forms of OA. Determinations of median lethal dose (LD50) were conducted by dosing day-old chicks and recording mortality for 10 days. LD50 values were 4.41, 3.95, and 2.69 mg/kg for OA acid, Na salt, and K salt, respectively. These results indicated that the K salt of OA was more toxic than the Na salt in acute oral dosing. During the feeding study, results also suggested that chemical form of OA affected its toxicity, but after feeding 3.0 micrograms/g OA for 4 weeks, no significant differences in toxicity were caused by the various chemical forms of OA or the A. ochraceus contaminated diet.

Comparative toxicity of Chaetomium contaminated corn and various chemical forms of oosporein in broiler chicks.

The toxicity to broiler chicks of Chaetomium contaminated corn and various chemical forms of oosporein were compared by feeding diets containing 60% Chaetomium contaminated corn (300 micrograms oosporein/g diet), and 300 or 150 micrograms/g of purified oosporein in either the K salt, Na salt, or organic acid form from hatching to 3 weeks of age. The Chaetomium contaminated corn diet caused 100% mortality during the first week of feeding. Necropsies revealed extensive visceral and articular gout, enlarged pale kidneys, dehydration, proventricular enlargement with mucosal necrosis, and a dark green discoloration of the gizzard lining. When the mortality percentages of the two experiments conducted were considered collectively, the K and Na salts of oosporein caused significantly higher mortality than the organic acid form of oosporein. The K salt caused the most severe lesions and the organic acid caused the least severe lesions. No mortality occurred at the 150 micrograms/g K salt or 150 micrograms/g organic acid levels. Relative kidney weights were increased by all forms of oosporein at 300 micrograms/g, but at 150 micrograms/g only the K salt caused an increase in kidney weight. The LD50 values, based on mortality from 1 to 10 days, were 5.77, 5.00, and 4.56 mg/kg for oosporein acid, oosporein Na salt, and oosporein K salt, respectively. These results suggest that the salts of oosporein (particularly the K salt) are more toxic than the organic acid, and the natural occurrence of oosporein in a salt form could contribute to the increased toxicity of the Chaetomium contaminated corn.