Hepatotoxicity of pulegone in rats: Its effects on microsomal enzymes, in vivo

Oral administration of pulegone (400 mg/kg) to rats once daily for five days caused significant decreases in the levels of liver microsomal cytochrome P-450 and heme. Cytochrome b5 and NAD(P)H-cytochrome c-reductase activities were not affected. Massive hepatotoxicy accompanied by an increase in serum glutamate pyruvate transaminase (SGPT) and a decrease in glucose-6-phosphatase were observed upon treatment with pulegone. A significant decrease in aminopyrine N-demethylase was also noticed after pulegone administration. Menthone or carvone (600 mg/kg), compounds related to pulegone, when administered orally did not cause any decrease in cytochrome P-450 levels. The hepatotoxic effects of pulegone were both dose and time dependent. Pretreatment of rats with phenobarbital (PB) or diethylmaleate (DEM) potentiated the hepatotoxicity caused by pulegone, whereas, pretreatment with 3-methylcholanthrene (3-MC) or piperonyl butoxide protected from it. It appears that a PB induced cytochrome P-450 catalysed reactive metabolite(s) may be responsible for the hepatotoxicity caused by pulegone.

Metabolism of a monoterpene ketone, R-(+)-pulegone—a hepatotoxin in rat

R-(+)-Pulegone was administered orally to rats and the urinary metabolites were investigated. Six metabolites were isolated and purified using column and thin layer chromatographic techniques. Metabolites were identified by i.r., n.m.r. and mass spectral analyses.The neutral metabolites isolated from urine of rats treated with pulegone (I) were: pulegol (II), 2-hydroxy-2(1'-hydroxy-1'-methylethyl)-5-methylcyclohexanone (III), 3,6-dimethyl-7a-hydroxy-5,6,7,7a-tetrahydro-2(4H)-benzofuranone (V) and menthofuran (VII). Metabolites II and III were also excreted in conjugated form.Acidic metabolites isolated from urine of rats treated with pulegone (I) were: 5-methyl-2(1'-methyl-1'-carboxyethylidene)cyclohexanone (IV) and 5-methyl-5-hydroxy-2(1'hydroxy-'-carboxyethyl)cyclohexanone (VI).

Biotransformations of α-terpineol in the rat: Its effects on the liver microsomal cytochrome P-450 system

Alpha-Terpineol (I), a monocyclic monoterpene tertiary alcohol, is widely used in the manufacture of perfumes, cosmetics, soaps and antiseptic agents. It was reported earlier (Horning et al. 1976) that this monoterpene alcohol when administered to humans is hydroxylated to p-menth-l,2,8-triol (II). It is not known whether c~-terpineol also produces other metabolites during its metabolism in the mammalian system and if so, the nature of these metabolites.

Effect of follicle-stimulating hormone and its antiserum on the activity of ornithine decarboxylase in the ovary of rat and hamster

The ability of FSH to stimulate the activity of ornithine decarboxylase (ODC) in the ovary of the immature rat and cycling hamster has been examined using specific antisera to gonadotropins. The stimulatory effect of FSH on ODC activity in the ovary of the immature rat was abolished when LH antiserum was administered along with FSH, while similar administration of FSH antiserum had no effect on LH action in stimulating ODC activity, thereby demonstrating the specificity of the LH effect. During the estrus cycle of the hamster, ODC activity in the ovary could be detected only on the evening of proestrus, the maximal activity seen at 1700 h being associated with both the Graafian follicles and the rest of the ovarian tissue. Neutralization of the proestrous FSH surge had no effect on the activity of ODC in either of these tissues, while similar administration of LH antiserum at 1300 h of proestrus completely inhibited the ODC activity in both large follicles and the rest of the ovarian tissue. Thus, the surge of LH, but not of FSH, appears to be responsible for regulating the ODC activity in the ovary of the cycling hamster.

Evidence for the formation of a known toxin, P-cresol, from menthofuran

Menthofuran (II, 4,5,6,7-tetrahydro-3,6-dimethyl benzofuran), the proximate toxin of R-(+)-pulegone (I), was administered orally to rats (200 mg/kg of body weight/day) for three days and the urinary metabolites were investigated. Among the several metabolites formed, two of them viz. 4-Hydroxy-4-methyl-2-cyclohexenone (VII) and p-cresol (VIII) were indentified. In support of the formation of these metabolites, it has been demonstrated that phenobarbital induced rat liver microsomes readily convert 4-methyl-2-cyclohexenone (V) to 4-hydroxy-4-methyl-2-cyclohexenone (VII) and p-cresol (VIII) in the presence of NADPH and O2. Possible mechanism for the formation of these two metabolites (VII, VIII) from menthofuran (II) has been proposed.

A Rapid Bioassay for Measuring Inhibin Activity

A sensitive and rapid bioassay of inhibin is described. Swiss albino female mice, 27 days old, weighing 25-30 g, were primed with 20 IU hCG, given in 2 equal doses, administered at 0900 h and 1800 h. An increment of 200% in uterine weight could be observed when the animals were autopsied the next day at 1000 h. The validity of the endpoint chosen, as a function dependent on FSH, was shown by the fact that neutralization of endogenous FSH by FSH antiserum led to an inhibition of uterine weight increase. Further injection of the inhibin material caused an actual reduction in the heightened levels of FSH (ng/ml) brought about by hCG: Control, 207 ± 13.6; hCG, 365 ± 28.8 and hCG inhibin, below the detection level of radioimmunoassay. Inhibin preparation from ovine testicular extract when tested at 3 dose levels showed a dose dependent and significant suppression in uterine weight increase. The assay was statistically validε = 2.5; slope = 23.9; λ = 0.104 and intra- and interassay variation = 8.3% and 11.7%, respectively. Since the mouse uterine weight assay is specific, has greater sensitivity and is uniformly reproducible, it is suggested that this assay procedure is ideally suited to assess the activity of different inhibin test preparations as well as to follow the purification of inhibin activity from crude material.

Oestrogen-induced synthesis of thiamin-binding protein in immature chicks. Kinetics of induction, hormonal specificity and modulation

A specific radioimmunoassay procedure was developed to monitor the plasma concentrations of thiamin-binding protein, a minor yolk constituent of the chicken egg. By using this sensitive assay, the kinetics of oestrogen-induced elaboration of this specific protein in immature chicks was investigated. After a single injection of the steroid hormone, with an initial lag period of 4–5h the thiamin-binding protein rapidly accumulated in the plasma, attaining peak concentrations around 75h and declining thereafter. A 4-fold amplification of the response was noticed during the secondary stimulation, and this increased to 9-fold during the tertiary stimulation with the steroid hormone. The magnitude of the response was dependent on the hormone dose, and the initial latent period and the duration of the ascending phase of induction were unchanged for the hormonal doses tested during both the primary and secondary stimulations. The circulatory half-life of the protein was 6h as calculated from the measurement of the rate of disappearance of the exogenously administered 125I-labelled protein. Simultaneous administration of progesterone, dihydrotestosterone or corticosterone did not alter the pattern of induction. On the other hand, hyperthyroidism markedly decreased the oestrogenic response, whereas propylthiouracil-induced hypothyroidism had the opposite effect. The anti-oestrogen E- and Z-clomiphene citrates, administered 30min before oestrogen, effectively blocked the hormonal induction. a-Amanitin and cycloheximide administered along with or shortly after the sex steroid severely curtailed the protein elaboration. A comparison of the kinetics of induction of thiamin- and riboflavin-binding proteins by oestrogen revealed that, beneath an apparent similarity, a clear-cut difference exists between the two vitamin-binding proteins, particularly with regard to hormonal dose-dependent sensitivity of induction and the half-life in circulation. The steroid-mediated elaboration of the two yolk proteins thus appears to be not strictly co-ordinated, despite several common regulatory features underlying their induction.

Acute toxicity of methyl isocyanate in mammals. II. Induction of hyperglycemia, lactic acidosis, uraemia, and hypothermia in rats

When rats were administered methyl isocyanate (MIC) by inhalation or subcutaneous route it produced severe hyperglycemia, clinical lactic acidosis, highly elevated plasma urea, and reduced plasma cholinesterase activity with unaltered erythrocytc acetyl cholinesterase activity. Irrespective of the route of administration, MIC also caused severe hypothermia, which was not ameliorated by prior administration of atropine sulphate. Acute toxic effects of MIC are essentially similar by either route except for the intensity of the effects

Acute toxicity of methyl isocyanate in rabbit: In vitro and in vivo effects on rabbit erythrocyte membrane

Methyl isocyanate (MIC) interaction with the rabbit erythrocyte membrane increased the fluidity of the membrane and decreased the osmotic fragility of erythrocytes both in vitro and in vivo in rabbits intoxicated with MIC subcutaneously. MIC inhibited both acetylcholinesterase (AChE) and adenosine triphosphatase (ATPase) activities of erythrocytes dose-dependently in vitro, while in vivo a decreased trend in ATPase activity with unaltered AChE activity was observed. MIC also caused significant decrease in plasma sodium level with corresponding increase in potassium level in rabbits. The observed effects are due to MIC, per se, as the hydrolysis products of MIC, methylamine and N,Nprime-dimethylurea did not affect the erythrocyte fluidity and enzymes activities both in vitro and in vivo while they increased the osmotic fragility of erythrocytes in vivo in rabbits administered subcutaneously in equimolar concentration to MIC dosage. Inhibition of Na+-K+-dependent ATPase with altered permeability to cations and also probably water transport of plasma membrane due to MIC interaction are envisaged.

Do the Hydrolysis Products, Methylamine and N,N'-Dimethylurea, Play any Role in the Methyl Isocyanate-Induced Haematological and Biochemical Changes in Rabbits?

The subcutaneous administration of methyl isocyanate (MIC) to female rabbits, resulted in significant increases in haemoglobin concentration, erythrocyte volume fraction and leucocyte number in blood, as well as plasma total proteins, and urea. The present study was designed to investigate whether the hydrolytic products of MIC, methylamine (MA) and N,N'-dimethylurea (DMU) play any role in eliciting these changes. Both MA and DMU administered subcutaneously in an equimolar dose to that of 1.0 LD50 MIC, 2.2 mmol kg-1, had no influence on these parameters, although there was a marginal increase in the plasma urea level shortly after the administration of DMU. This study establishes that the observed haematological and biochemical changes induced by MIC intoxication in rabbits are mostly due to MIC.

Influence of methylamine and N,N'-dimethylurea, the hydrolysis products of methyl isocyanate, on its systemic toxicity

Subcutaneous administration of the LD50 dose of methyl isocyanate (MIC) to rats induced severe hyperglycaemia, lactic acidosis and uraemia in rats. Neither methylamine (MA) nor N,N′-dimethylurea (DMU), the hydrolysis products of MIC, administered in equimolar doses had any influence on these parameters except for a marginal transient increase in plasma urea by DMU. Methyl isocyanate administration led to haemoconcentration, resulting in an increase in the plasma concentration of total proteins and a decrease in both the plasma concentration of albumin and the plasma cholinesterase activity. The hydrolysis products of MIC had no influence on any of these parameters. Thus, it seems reasonable to suggest that the systemic effects of MIC are caused by MIC per se, in spite of its high hydrolytic instability.

Augmentation of murine natural killer cell and antibody dependent cellular cytotoxicity activities by phyllanthus emblica, a new immunomodulator

When administered orally, Phyllanthus emblica, an excellent source of vitamin C (ascorbate), has been found to enhance natural killer (NK) cell activity and antibody dependent cellular cytotoxicity (ADCC) in syngeneic BALB/c mice, bearing Dalton's lymphoma ascites (DLA) tumor. P. emblica elicited a 2-fold increase in splenic NK cell activity on day 3 post tumor inoculation. Enhanced activity was highly significant on days 3, 5, 7 and 9 after tumor inoculation with respect to the untreated tumor bearing control. A significant enhancement in ADCC was documented on days 3, 7, 9, 11 and 13 in drug treated mice as compared to the control. An increase in life span (ILS) of 35% was recorded in tumor bearing mice treated with P. emblica. This increased survival was completely abrogated when NK cell and killer (K) cell activities were depleted either by cyclophosphamide or anti-asialo-GM, antibody treatment. These results indicate: (a) an absolute requirement for a functional NK cell or K cell population in order that P. emblica can exert its effect on tumor bearing animals, and (b) the antitumor activity of P. emblica is mediated primarily through the ability of the drug to augment natural cell mediated cytotoxicity.

Effect of blocking oestrogen synthesis with a new generation aromatase inhibitor CGS 16949A on follicular maturation induced by pregnant mare serum gonadotrophin in the immature rat

While the endocrine role of oestrogen is well established, its function in follicular maturation as an autocrine or paracrine regulator is less well understood. This study was designed to delineate the requirement of oestrogen for follicular development in immature rats. Exogenous gonadotrophin (25 IU pregnant mare serum gonadotrophin (PMSG) per rat) was administered to 21- to 23-day old female rats to induce follicular growth and development. In the experimental animals, synthesis of oestrogen was blocked by implanting an Alzet pump containing the aromatase inhibitor (AI) CGS 16949A (fadrozole hydrochloride; 50 mu g/rat per day). The treatment resulted in blockade of the PMSG induced increase in both serum and intrafollicular oestrogen (>95%), thus leading to an inhibition in uterine weight increment. Compared with the controls, ovarian weight increased markedly in both the PMSG (295%)- and PMSG+AI (216%)-primed animals. There was no significant difference in either the proliferative capabilities of the ovarian granulosa cells or their responsiveness to human chorionic gonadotrophin (hCG; 200 pg/ml) and ovine FSH (20 ng/ml) between the PMSG- and PMSG+AI-treated groups. Histological examination of the ovary, however, indicated a decrease in the number of healthy antral follicles in the Al-treated group compared with the PMSG-primed animals but both the groups showed a percentage increase over the controls (PMSG, 225; PMSG+AI, 158). The responsiveness of the animals to an ovulatory dose of hCG was drastically reduced (>80% inhibition of ovulation) in the oestrogen-deprived animals; this could be overriden by exogenous administration of oestrogen. In conclusion, although blocking oestrogen synthesis in the PMSG-primed rat does not seem to alter the functional properties of the isolated granulosa cells in vitro there appears to be an effect on the number of follicles which complete maturation and are able to ovulate in vivo.

In vivo and in vitro studies on the differential role of luteinizing hormone and follicle-stimulating hormone in regulating follicular function in the bonnet monkey (Macaca radiata) using specific gonadotropin antibodies

Although a distinct need for FSH in the regulation of follicular maturation in the primate is well recognized, it is not clear how FSH controls the functionality of different cellular compartments of the follicle. It is also not evident whether there is a requirement for LH in follicular maturation in the primate. In the first part of the present study, female bonnet monkeys were administered a well-characterized ovine (o) LH antiserum to neutralize endogenous monkey LH for different periods during the follicular phase, and the effect on the overall follicular maturation process was assessed by analyzing serum estrogen (E) and progesterone (P) profiles. Neither continuous LH deprivation from Day 8 of the cycle nor deprivation of LH on any one day between Days 6 and 10 had a significant effect on serum E and P profiles and the follicular maturation process. The period for which the antiserum was effective was dependent upon the dose injected; 1 ml of the antiserum given on Day 8 blocked ovulation but not follicular maturation. To assess the effect of deprivation of LH/FSH at the cellular level, animals were deprived in vivo of LH (on Days 8 and 9 of the cycle) or of FSH (on Day 6 of the cycle) by injection of highly characterized hCG and ovine (o) FSH antisera, respectively; the in vitro responsiveness of granulosa and thecal cells isolated on Day 10 from these animals was then determined.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro and in vivo effect of methyl isocyanate on rat liver mitochondrial respiration

Previous work has shown that irrespective of the route of exposure methyl isocyanate (MIC) caused acute lactic acidosis in rats (Jeevaratnam et al., Arch. Environ. Contam. Toxicol. 19, 314�319, 1990) and the hypoxia was of stagnant type due to tissue hypoperfusion resulting from hypovolemic hypotension in rabbits administered MIC subcutaneously (Jeevarathinam et al., Toxicology 51, 223�240, 1988). The present study was designed to investigate whether MIC could induce histotoxic hypoxia through its effects on mitochondrial respiration. Male Wistar rats were used for liver mitochondrial and submitochondrial particle (SMP) preparation. Addition of MIC to tightly coupled mitochondria in vitro resulted in stimulation of state 4 respiration, abolition of respiratory control, decrease in ADP/O ratio, and inhibition of state 3 oxidation. The oxidation of NAD+-linked substrates (glutamate + malate) was more sensitive (fiveto sixfold) to the inhibitory action of MIC than succinate while cytochrome oxidase remained unaffected. MIC induced twofold delay in the onset of anerobiosis, and cytochrome b reduction in SMP with NADH in vitro confirms inhibition of electron transport at complex I region. MIC also stimulated the ATPase activity in tightly coupled mitochondria while lipid peroxidation remained unaffected. As its hydrolysis products, methylamine and N,N?-dimethylurea failed to elicit any change in vitro; these effects reveal that MIC per se acts as an inhibitor of electron transport and a weak uncoupler. Administration of MIC sc at lethal dose caused a similar change only with NAD+-linked substrates, reflecting impairment of mitochondrial respiration at complex I region and thereby induction of histotoxic hypoxia in vivo.