The direct toxicity of alpha-naphthylisothiocyanate in cell culture

α-naphthylisothiocyanate (ANIT) kills rat liver cells in culture. Testing of a variety of related compounds revealed that toxicity depended upon the isothiocyanate group. The toxicity of ANIT was similar on several lines of cultured cells and in the presence of inhibitors and inducers of microsomal enzyme systems. The addition of a microsomal metabolizing system from rat liver to the cell cultures greatly enhanced the toxicity of 2 hepatotoxins requiring metabolism, but did not affect the toxicity of ANIT. It is concluded therefore that ANIT is toxic without biotransformation.     

Influence of adrenergic nervous and prostaglandin systems on hydralazine-induced renin release

The role of the beta-adrenergic nervous and prostaglandin systems in vasodilator-induced activation of the renin-angiotensin system was studied in conscious rats. The plasma renin activity (PRA) response to intravenous hydralazine (0.25, 0.5 and 1 mg/kg body wt.) was compared to the PRA response following administration of similar doses of hydralazine to rats pretreated with either indomethacin (3 mg/kg body wt. i.v.) or indomethacin and propranolol (1 mg/kg body wt. i.v.). PRA increased significantly above control levels after each of the hydralazine doses. In rats pretreated with indomethacin, PRA did not increase with the 0.25 mg/kg dose of hydralazine; increased significantly with the 0.5 mg/kg dose but remained significantly lower than the PRA response in the absence of indomethacin; and increased with the 1 mg/kg dose to a level not significantly different from PRA in rats receiving only hydralazine. When rats were pretreated with indomethacin and propranolol, PRA did not increase significantly in response to either the 0.25 or 0.5 mg/kg doses of hydralazine. Although a statistically significant increase in PRA was noted with the 1 mg/kg dose of hydralazine, the level of PRA achieved was very low and only 15% of that observed with the other two treatment regimens (i.e., hydralazine alone or indomethacin and hydralazine). These results demonstrate that vasodilator-induced renin release is only partially mediated via the prostaglandin system, that the degree of this control is related to the intensity of vasodilator stimulus and that renin release following administration of hydralazine can be attributed almost entirely to activation of the beta-adrenergic nervous and prostaglandin systems.     

Biosynthesis of cholic acid in rat liver. 24-Hydroxylation of 3alpha, 7alpha, 12alpha-trihydroxy-5beta-cholestanoic acid.

Conversion of 3alpha, 7alpha, 12alpha-trihydroxy-5beta-[7beta-3H]cholestanoic acid into 3alpha, 7alpha, 12alpha, 24-tetrahydroxy-5beta-cholestanoic acid in rat liver was catalyzed either by the mitochondrial fraction fortified with the 100,000 times g supernatant fluid or the microsomal fraction fortified with 100,000 times g supernatant fluid and ATP. The microsomal system was more active than the mitochondrial system. With the microsomal system the rate of reaction was considerably faster with free 3alpha, 7alpha, 12alpha-trihydroxy-5beta-cholestanoic acid as substrate than with the corresponding coenzyme A ester. Addition of coenzyme A inhibited the activity. Addition of cofactors other than ATP and coenzyme A did not markedly influence the reaction. The 100,000 times g supernatant fluid could be substituted with a protein fraction obtained by ammonium sulfate precipitation and Sephadex chromatography of the 100,000 times g supernatant fluid. The reaction was not catalyzed by a mixed function oxidase since there was no incorporation of 18O into the product when the reaction was performed in an atmosphere containing 18O2. On the other hand, oxygen may be obligatory since there was almost complete inhibition when the reaction was performed in an atmosphere consisting of nitrogen. Carbon monoxide did not inhibit the reaction. One atom of deuterium was incorporated into the product when the reaction was performed in a medium containing deuterated water. It was concluded that microsomal 24-hydroxylation of 3alpha, 7alpha, 12alpha-trihydroxy-5beta-cholestanoic acid involves the combined action of a desaturase and a hydratase. The reaction catalyzed by the hydratase appears to be stereospecific since the 24alpha epimer of 3alpha, 7alpha,12alpha-trihydroxy-5beta-cholestanoic acid was the predominant product. In contrast to the microsomal system, the mitochondrial system was not stimulated by the addition of ATP and was not inhibited by coenzyme A. The coenzyme A ester of 3alpha, 7alpha, 12alpha-trihydroxy-5beta-cholestanoic acid was 24-hydroxylated more efficiently than the free acid.     

Acetaminophen toxicity in lymphocytes heterozygous for glutathione synthetase deficiency

We have studied the effects of acetaminophen metabolites generated by a murine hepatic microsomal system on lymphocytes from two subjects heterozygous for glutathione synthetase deficiency. Heterozygous cells exhibited greater dose-related toxicity than controls. Following a 2-h incubation with acetaminophen and the microsomal system, cells were washed and incubated for 16 h in the presence or absence of N-acetylcysteine, the standard antidote for acetaminophen toxicity. In control cells, glutathione content was replenished to nearly base-line values and toxicity was prevented. N-Acetylcysteine thus prevented toxicity even after covalent binding of acetaminophen metabolites had occurred. Heterozygous cells failed to use N-acetylcysteine as efficiently to resynthesize glutathione, and the cells were not protected from acetaminophen toxicity. Heterozygotes may be at increased risk of toxicity from drugs whose metabolites are detoxified by glutathione conjugation.     

Toxicological Evaluation of Lactase Derived from Recombinant Pichia pastoris

A recombinant lactase was expressed in Pichia pastoris, resulting in enzymatic activity of 3600 U/mL in a 5 L fermenter. The lactase product was subjected to a series of toxicological tests to determine its safety for use as an enzyme preparation in the dairy industry. This recombinant lactase had the highest activity of all recombinant strains reported thus far. Acute oral toxicity, mutagenicity, genotoxic, and subchronic toxicity tests performed in rats and mice showed no death in any groups. The lethal dose 50% (LD₅₀) based on the acute oral toxicity study is greater than 30 mL/kg body weight, which is in accordance with the 1500 L milk consumption of a 50 kg human daily. The lactase showed no mutagenic activity in the Ames test or a mouse sperm abnormality test at levels of up to 5 mg/plate and 1250 mg/kg body weight, respectively. It also showed no genetic toxicology in a bone marrow cell micronucleus test at levels of up to 1250 mg/kg body weight. A 90-day subchronic repeated toxicity study via the diet with lactase levels up to 1646 mg/kg (1000-fold greater than the mean human exposure) did not show any treatment-related significant toxicological effects on body weight, food consumption, organ weights, hematological and clinical chemistry, or histopathology compared to the control groups. This toxicological evaluation system is comprehensive and can be used in the safety evaluation of other enzyme preparations. The lactase showed no acute, mutagenic, genetic, or subchronic toxicity under our evaluation system.     

Drug-induced antinuclear antibodies in the guinea pig

Antinuclear antibodies (ANA) development was studied in male guinea pigs in response to chronic treatment with procainamide, hydralazine, acetanilide or caffeine. Acetanilide and caffeine have not previously been associated with ANA induction. Fifty-one weanling Hartley guinea pigs were divided into five groups which received either procainamide, hydralazine, acetanilide, caffeine or saline sc for 55 weeks; drug dosage was 10 mg/kg initially and was increased incrementally to 40 mg/kg by 10 months except for hydralazine, which was increased to 20 mg/kg. Two weeks before initiation of treatment, 1 mg of the appropriate drug in 0.4 ml of buffered Freund's complete adjuvant (FCA-PBS) was administered intradermally. Controls received FCA-PBS only. Sera ANA were assayed at 6, 10 and 13 months. After 13 months of treatment, those sera which were ANA positive were assayed for anti-deoxyribunucleoprotein antibodies and were titered for ANA. Chi-square analyses were performed on results of the 10- and 13-month ANA screening results. ANA induction was significant at P = 0.05 only for the group receiving procainamide at both 10 and 13 months of treatment. When the cumulative results of all ANA screens were analyzed, ANA induction was significant for procainamide, acetanilide and caffeine. The test system did not prove to be promising for unambiguous identification of drugs with ANA-inducing potential, but may be useful for studies of mechanisms of ANA induction by chemicals.     

Some properties of the fatty alcohol oxidation system and reconstitution of microsomal oxidation activity in intestinal mucosa

This paper describes the metabolism of fatty alcohols by microsomal and cytosolic fractions from intestinal mucosa. Microsomes of rabbit intestinal mucosa had a high activity of [1-14C]dodecanol oxidation as did those of liver. The intestinal cytosolic fraction also exhibited oxidation activity to a lesser extent than the microsomes did. The reaction product was determined as lauric acid using thin-layer chromatography. Laurylaldehyde was detected as another product, when semicarbazide was added to the incubation system. Cyclodextrins exhibited a stimulation effect similarly to bovine serum albumin on the microsomal activity. We have compared the stimulatory effects of dimethyl-beta-cyclodextrin, beta-cyclodextrin, gamma-cyclodextrin and alpha-cyclodextrin, which decrease in that order. Effects of NAD+ and dodecanol concentrations, pH and pyrazole on microsomal activity were compared with those on cytosolic activity. Dodecanol oxidation activity was solubilized and reconstituted with a fatty alcohol dehydrogenase and a fatty aldehyde dehydrogenase separated from the intestinal microsomes. These findings indicate that both the dehydrogenases participate in microsomal oxidation of fatty alcohols to fatty acids with fatty aldehydes as intermediates in the reaction.     

The metabolism and binding of catecholamines by the hepatic microsomal mixed-function oxidase of the rat.

Noradrenaline and adrenaline were metabolized by an NADPH- and oxygen-dependent process located within the hepatic microsomal fraction of the rat. Metabolism was inhibited by CO and compound SKF 525A, but not by pargyline, an inhibitor of monoamine oxidase, or by 3,4-dimethoxy-5-hydroxybenzoic acid, an inhibitor of catechol O-methyltransferase. It is concluded that the enzyme system responsible for the metabolism of the catecholamines was the microsomal mixed-function oxidase. The Km for noradrenaline was 2.4 mM and for adrenaline 1.0 mM, and V 15.6 and 3.6 nmol/min per mg of microsomal protein respectively. Both catecholamines bound to the microsomal fraction, producing a type II spectral change, with a Ks for noradrenaline of 0.9 mM and for adrenaline of 1.0 mM, and showed other characteristics of type II compounds by inhibited the reduction of cytochrome P-450 by NADPH and exhibiting an enhanced metabolism in the presence of acetone. The major product of catecholamine metabolism was an as yet unidentified alkali-labile compound, which did not correspond to any of the recognized catecholamine metabolites.     

Effects of hydralazine on guanosine cyclic 3', 5'-monophosphate levels in rat aorta

The mechanism of the vasodilator effect of hydralazine on isolated rat aorta was studied. Results demonstrated that the vasodilator effect of hydralazine was greater on intact aortas than on endothelium-denuded preparations, particularly at low concentrations of between 0.1 mM and 0.5 mM. In addition, hydralazine did not have any effect on cyclic GMP levels. We also found that methylene blue, an inhibitor of guanylate cyclase, completely abolished the vasorelaxant action of nitroglycerin but not that of hydralazine. These results indicate that the vasodilator effect of hydralazine was not due to elevating the cyclic GMP levels. On the other hand, hydralazine significantly inhibited both the contractions induced by norepinephrine and/or high-potassium. In conclusion, a part of the vasodilator effect of hydralazine seems to depend on the integrity of the vascular endothelium. However, this vasodilator effect was not associated with any elevation in cyclic GMP level. Thus, the direct vasodilator action of hydralazine may be related to its interference with the movement and/or translocation of calcium across the cell membrane.     

Hydralazine,antinuclear antibodies,and the lupus syndrome.

The incidence of patients with positive antinuclear antibody test results rose during three years of treatment with hydralazine. At the end of that period over half of the patients (both rapid and slow acetylators) had titres exceeding 1/20, but the rate of rise was faster in the slow acetylators than in the rapid. There was a significant relation between the cumulative dose of hydralazine and the proportion of patients found to have antinuclear factors. Fewer black patients had positive test results than white. Patients whose antinuclear antibody test results changed fron negative to positive during the study showed this change five to 26 months after beginning treatment. Some patients showed a substantial fall in antinuclear antibody titre even though hydralazine was continued. From these findings patients in whom test results for antinuclear antibody became positive during treatment with hydralazine need not have the drug stopped unless they have clinical features of the lupus syndrome.     

Prostaglandin generation from gastroduodenal mucosa: regional and species differences

Regional and species differences in prostaglandin synthesis from gastroduodenal mucosa were assessed radiometrically. In the presence of excess added arachidonic acid substrate, corporal mucosa generated more prostanoid product per DNA than did antral or duodenal mucosa whether the whole homogenate or the microsomal fraction was used as an enzyme source. This appeared to be secondary to variability in cyclooxygenase activity and could not be explained by regional differences in the activity of enzymes competing for arachidonic acid substrate, in free endogenous arachidonic acid levels, in prostaglandin catabolizing activity, or in homogenate inhibitors. The qualitative product profile differed between species but not between regions within a species.     

Accelerated microsomal DNA synthesis under the influence of xenobiotics and chemical carcinogens]

Injection of 3,4-benz(a)pyrene, methyl nitrosourea and phenobarbital into healthy mice of the C3HA line results in a rapid, sharp increase of [14C]-thymidine incorporated into liver microsomal DNA, accompanied by a suppression of nuclear DNA synthesis. In the liver of neoplastic mice and in the ascite cells of hepatoma 22A the system of microsomal DNA synthesis was insensitive to the injection of methyl nitrosourea. Cycloheximide and puromycin, which strongly inhibited nuclear DNA synthesis, had no effect on the synthesis of microsomal DNA. Stimulation of [14C]-thymidine incorporation into microsomal DNA after injection of methyl nitrosourea and 3,4-benz(a)pyrene may be accounted for not only by an increase of the DNA reparation processes, since caffeine, the inhibitor of post-replicatory reparation of DNA, did not eliminate the induction of microsomal DNA synthesis in the liver. Hydroxyurea in combination with methyl nitrosourea and phenobarbital significantly suppressed the synthesis of nuclear DNA in the liver and did not affect the synthesis of mtDNA; the stimulating effects of these inducers on the synthesis of microsomal DNA was thereby removed. This is indicative of independence of synthesis of microsomal DNA on that of nuclear DNA and mtDNA. Different specific radioactivities of microsomal, nuclear and mtDNAs in the regenerating mouse liver on the 5th, 10th and 15th post-hepatectomy days may be due to different metabolic stability of these DNAs. A possible role of microsomal DNA as a xenobiotic system component is discussed.     

Hydralazine stimulates production of oxygen free radicals in Eagle's medium and cultured fibroblasts.

The effect of hydralazine on the oxygen free radical production was studied in whole cultured murine liver fibroblasts and mitochondrial and microsomal fractions of the cells by ESR spin trapping with DMPO and measurement of Tiron semiquinone formation. Hydralazine itself was found to generate free radicals in phosphate buffer and especially in Eagle's Minimal Essential Medium. Most of the adduct of the spin trap DMPO was due to its reaction with hydralazine-induced hydroxyl radical. Moreover, this compound stimulated free radical formation in fibroblasts. These data suggest that hydralazine alters the cellular free radical metabolism which may have implications for the biological activity of this drug.     

Irreversible protein binding of norethisterone (norethindrone) epoxide.

14,15-3H-Norethisterone-4 beta, 5 beta-epoxide, a metabolite of norethisterone, was incubated with several proteins and nucleic acids. After 30 min incubation 0.19 nmol of the epoxide were irreversibly bound per mg albumin which contains free sulfhydryl groups; proteins without SH-groups, such as concanavalin A, gamma-globulin, DNA and RNA, did not irreversibly bind norethisterone epoxide. A superoxide (O2) generating enzyme system comprised of xanthine oxidase and hypoxanthine was capable of catalyzing the irreversible binding of the parent compound, norethisterone, to albumin, indicating that an oxidation product was formed which reacted with the protein. When norethisterone epoxide was incubated for 60 min with hepatic microsomes of rats in absence of NADPH, about 2.0 nmol of the epoxide were irreversibly incorporated per mg microsomal protein. This binding was increased to 5.2 nmol by addition of a NADPH regenerating system. Addition of glutathione and cytosol decreased only the NADPH-dependent protein binding; phenobarbital pretreatment of rats induced this NADPH-dependent binding of norethisterone epoxide to microsomal protein by a factor of 2. In presence of NADPH, binding of the epoxide to microsomal protein depended on substrate concentration used. The results indicate that norethisterone epoxide is able to chemically react with proteins. In addition, hepatic microsomal enzymes convert the epoxide to another metabolite which also can react with proteins.     

Changes in angiotensin receptors expression play a pivotal role in the renal damage observed in spontaneously hypertensive rats

The renal renin-angiotensin system plays a central role in the development of hypertension. The aim of this work was to verify the expression of angiotensin II receptors AT(1)R and AT(2)R in the microsomal fraction of renal cortex and correlate this with the development of hypertension and renal damage in spontaneously hypertensive rats (SHR) using Wistar-Kyoto rats (WKY) as controls. AT(1)R expression increased (126%) and AT(2)R expression decreased (66%) in 4-wk-old SHR; AT(2) expression decreased in 14-wk-old SHR (61%) compared with respective age-matched WKY. These modifications were correlated to the increase in protein kinase C activity and decrease in protein kinase A activity. Four-week-old SHR showed large accumulations of macrophages in kidney glomerulus and the tubulointerstitial area, dense cortical collagen deposition, and arterial proliferative changes in the walls of arterioles and medium-sized vessels. Similar modifications were also observed in 14-wk-old SHR. Four-week-old SHR treated with losartan (30 mg·kg(-1)·day(-1)) or hydralazine (15 and 30 mg·kg(-1)·day(-1)) by gavage for 10 wk did not develop hypertension. The decrease in AT(2)R expression and renal damage observed in SHR remained even after treatment with hydralazine. On the other hand, losartan treatment prevented the modifications observed in 14-wk-old SHR, indicating that renal injuries are caused specifically by AT(1) rather than an increase in blood pressure. Our results indicate that the imbalance in AT(1)R and AT(2)R expression is associated with an inflammatory process that contributes to renal injury in adult SHR and to the development of hypertension.     

Inhibitory effect of propylthiouracil-induced hypothyroidism in rat on oxidative drug metabolism

The effect of propylthiouracil (PTU) pretreatment on in vivo and in vitro oxidative drug metabolism was determined in the rat. Whereas pentobarbital sleeping time (PBST) and zoxazolamine paralysis time (ZZPT) were used as indices of in vivo drug metabolizing activity, biotransformation of aminopyrine and aniline by hepatic microsomal preparations were used as indices of in vitro drug metabolizing enzymes activities. PTU pretreatment significantly prolonged both PBST and ZZPT. Whereas PTU did not affect microsomal protein concentration or cytochrome P-450 content, it significantly decreased microsomal cytochrome c reductase and aniline hydroxylase activities. These changes in enzymatic activities were observed in microsomal preparations from either non-fasted or 24-hr fasted rats. Our results suggest that PTU-induced hypothyroidism modifies the metabolism and effectiveness or toxicity of concomitantly administered drugs.     

Systemic vasodilatation and renal sodium excretion: Effects of hydralazine and diazoxide

The effects on renal sodium excretion of two systemic vasodilators, hydralazine and diazoxide, were investigated in volume expanded, anesthetized rats with unilaterally denervated kidneys. Urinary sodium excretion and fractional excretion of filtered sodium increased following hydralazine but decreased following diazoxide. Changes in renal hemodynamics were dissimilar as well: renal plasma flow was increased following hydralazine, but unchanged with diazoxide. All changes in renal sodium excretion and renal hemodynamics following hydralazine were prevented by pretreatment with indomethacin. Renal denervation accentuated the increases in fractional sodium excretion and renal blood flow that occured following hydralazine.Hydralazine and diazoxide differ substantially in their effects on renal sodium excretion, apparently due to the stimulation of renal prostaglandins by the former agent. Although renal innervation attenuates the natriuretic effect of hydralazine, stimulation of the sympathetic nervous system does not account for differences in the renal effects of these two drugs.     

Crude oil effects on microsomal mixed-function oxidase system components in the striped mullet (Mugil cephalus)

The effects of exposure to two types of crude oil on microsomal mixed-function oxidase system components in livers of juvenile striped mullet ($\text{Mugil cephalus}$) were investigated. Mullet were exposed for 4 days to emulsified Empire Mix or Saudi Arabian crude oils at an initial concentration of 75 ppm and an average of 1 ppm in the water column. Liver size was increased by about 50% following exposure to both oils. Since neither total hepatic protein nor microsomal protein increased as rapidly as did liver size, the concentrations of both were reduced following oil exposures. The proportion of microsomal protein to total hepatic protein or wet weight was not altered following crude oil exposure. Both cytochromes P-450 and $\text{b}$₅ were induced following oil treatment. NADPH-dependent enzymes assayed with cytochrome $\text{c}$ and dichlorophenolindophenol as substrates showed increases in activity after exposure to Empire Mix crude oil but only the latter enzyme activity was increased on a microsomal protein basis following Saudi Arabian crude oil treatment. Activities of NADH cytochrome $\text{c}$ and NADH cytochrome $\text{b}$₅ reductases appeared to vary with the protein level. However, since liver size was increased, oil-treated mullet had more of all parameters measured than did control mullet. Although the acute toxicity of Saudi Arabian crude oil to mullet is greater than that of Empire Mix crude oil, Empire Mix crude oil had greater inductive effects on microsomal oxidase components.     

Development of a new method for the preparation of an acellular allodermis,quality control and cytotoxicity testing

Demand for use of acellular allodermis is high but commercially appropriate products are not used routinely because of very high price and limited availability. These facts did motivate us to prepare acellular allodermis using a new, simple and less expensive method. We have developed a original method for preparation of acellular allogeneic dermis based on action of a proteolytic enzyme in combination with distilled water. Hypotonic environment in comparison with SDS or Triton ansure no toxicity of the final product. Trials for determination of optimal trypsin concentrations, temperature and time of action were performed. According to our results, the use of 2.5% trypsin/EDTA solution overnight at +4 °C was proving to be optimal. The histology confirmed absence of cells in the prepared dermis. No toxicity of final acellular dermis was confirmed by three independent tests (agar diffusion test contact cytotoxicity test and grow curve). The prepared acellular dermis seems to be suitable not only for direct clinical use, but it can be used as a scaffold for cell cultivation as well.     

Inhibition of hepatic glucose 6-phosphatase system by the green tea flavanol epigallocatechin gallate

Effect of 5-100 microM epigallocatechin gallate (EGCG) on hepatic glucose 6-phosphatase (G6Pase) system was investigated. EGCG inhibited G6Pase in intact but not in permeabilized rat liver microsomes, suggesting the interference with the transport. However, EGCG did not hinder microsomal glucose 6-phosphate (G6P) uptake. Instead, it increased the accumulation of radioactivity after the addition of [(14)C]G6P, presumably due to a slower release of [(14)C]glucose, the product of luminal hydrolysis. Indeed, EGCG was found to inhibit microsomal glucose efflux. Since G6Pase activity is depressed by glucose in a concentration-dependent manner, we concluded that EGCG inhibits G6Pase through an elevated luminal glucose level.