Clearance of N-nitrosodimethylamine and N-nitrosodiethylamine by the perfused rat liver. Relationship to the Km and Vmax for nitrosamine metabolism

The first-pass clearance of dietary N-nitrosodimethylamine (NDMA) by the liver is the most important factor in the pharmacokinetics of this carcinogen in the rat, but is less important in the pharmacokinetics of N-nitrosodiethylamine (NDEA). The reason for the difference in clearance of these two nitrosamines is not known. These experiments were carried out to see whether the general characteristics of the clearance of these two carcinogens in vivo could be reproduced in the perfused liver, and whether the clearance could be correlated with the Michaelis-Menten parameters Km and Vmax for their metabolism. If this could be done one would be able to predict the possible extent of first-pass clearance of nitrosamines in man from measurement of Km and Vmax for nitrosamine metabolism by the human liver. The Km (22 microM) and Vmax (10.2 and 13.4 nmol/g liver/min) for the metabolism of NDMA by slices from two human livers, the inhibition of that metabolism by ethanol (Ki 0.5 microM), and the rate of N-7 methylation of DNA when slices are incubated with NDMA, were measured. These results are similar to those reported previously with rat liver. The Km (27 microM) for the metabolism of NDEA by rat liver slices and the inhibition of that metabolism by ethanol (Ki 1 microM) were estimated from the rate of ethylation of the DNA of the slices. The clearance of both these nitrosamines by the perfused rat liver was measured, and the results appeared to parallel those in vivo with a striking difference between the clearance of NDMA and NDEA. The maximal rate of clearance of NDMA was 11.2 nmol/g liver/min and of NDEA 8.9 nmol/g liver/min, similar to the Vmax for metabolism of NDMA by liver slices and to the estimated maximal rate of liver metabolism of both nitrosamines in the living rat. However, although the Km for metabolism of these two nitrosamines by liver slices is similar (about 25 microM), the logarithmic mean sinusoidal concentration [see Bass and Keiding, Biochem Pharmacol 37: 1425-1431, 1988] giving half maximal clearance during perfusion (the equivalent to Km) was 2.3 microM for NDMA and 10.6 microM for NDEA. The almost 5-fold difference between these two values is the basis for the difference between the clearance of the two nitrosamines.(ABSTRACT TRUNCATED AT 400 WORDS)     

In vivo aspects of progesterone distribution and metabolism

The end-organ response of any hormone is the result of many factors which precede the event, including biosynthesis, secretion, transport, distribution, and metabolism. These factors vary among different species. The metabolic clearance rate (MCR) of progesterone varies between 40 and 180 L./day/Kg. in man (60 to 70), monkey (40 to 50), rabbit (55 to 60), sheep (110), rat (120), and guinea pig (180). Major sites of clearance include liver, brain, and uterus. Specific metabolites of progesterone include 20 alpha-hydroxypregn-4-en-3-one (20 alphaOHP) and alpha-pregnan-3,20-dione (5 alpha-DPH). Liver, brain, and uterine clearances, extractions, and conversions of progesterone to these metabolites have been studied in various species under apparent steady-state conditions. A specific hormone action of progesterone, the appearance of uteroglobin in the rabbit uterus, has also been studied in varying horomonal states (estrogen, estrogen plus progesterone, and progesterone alone). These have all been used as examples of progesterone distribution and metabolism.     

The effects of experimental malnutrition on albumin metabolism and distribution in rabbits

In order to determine the extent to which the concentration of albumin in plasma is maintained at the expense of the extravascular pool during protein-energy malnutrition, the rates of exchange between albumin in plasma (IA) and the extravascular pool (EA) and consequently the distribution of albumin between intravascular and extravascular pools (expressed as EA:IA) were measured in protein-energy-depleted and control rabbits. The fractional rates of synthesis (FSR) and catabolism (FCR), the concentration of albumin and the plasma volume (PV) were also measured. In animals in which protein-energy intake was reduced by 58% the concentration of albumin in plasma remained unchanged, whereas FCR decreased by 38% and FSR by 30%. No significant changes in EA:IA or PV were found. We conclude that albumin concentration during protein-energy depletion is not maintained at the expense of extravascular albumin, but by parallel changes in the rates of catabolism and synthesis.     

Uptake, distribution and metabolism of isoprenaline in the dog saphenous vein

Morphine withdrawal was precipitated by injection of various morphine antagonists into restricted parts of the ventricular system or by microinjection of levallorphan into specific brain areas of rats made dependent on morphine by repeated pellet implantation. When the antagonists could spread only within the lateral ventricles and the 3rd ventricle, a weak withdrawal syndrome was induced; by antagonist administration into the restricted 4th ventricle, however, strong withdrawal signs like jumping were elicited even at small dosages. In microinjection experiments, structures in the midbrain and the lower brain stem proved to be the most sensitive to antagonist action. Although microinjections into thalamic nuclei also had some effect, it could not be excluded that the effects were due to uncontrolled spreading of the drug. This became especially clear from experiments with tritium-labeled levallorphan. It is concluded that brain structures located in the anterior parts of the floor of the 4th ventricle and/or caudal parts of the periaqueductal gray matter are important sites of action for the development of physical dependence on morphine.     

In vitro metabolism of nicotine in liver of ageing mice

14-C-nicotine was incubated with 10000 X g supernatant fraction of liver homogenate from female mice (C3H/TIF/BOM) 2, 12 and 18 months old. The rate of metabolism was measured by the determination of the oxidative nicotine metabolite continine and by determining the disappearance of nicotine. No significant changes between different ages were found either in the continine formation or the rate of disappearance of nicotine.     

Acute stimulation of glucose metabolism in mice by leptin treatment

Leptin is an adipocyte hormone that functions as an afferent signal in a negative feedback loop regulating body weight, and acts by interacting with a receptor in the hypothalamus and other tissues. Leptin treatment has potent effects on lipid metabolism, and leads to a large, specific reduction of adipose tissue mass after several days. Here we show that leptin also acts acutely to increase glucose metabolism, although studies of leptin's effect on glucose metabolism have typically been confounded by the weight-reducing actions of leptin treatment, which by itself could affect glucose homoeostasis. We have demonstrated acute in vivo effects of intravenous and intracerebroventricular administrations of leptin on glucose metabolism. A five-hour intravenous infusion of leptin into wild-type mice increased glucose turnover and glucose uptake, but decreased hepatic glycogen content. The plasma levels of insulin and glucose did not change. Similar effects were observed after both intravenous and intracerebroventricular infusion of leptin, suggesting that effects of leptin on glucose metabolism are mediated by the central nervous system (CNS). These data indicate that leptin induces a complex metabolic response with effects on glucose as well as lipid metabolism. This response is unique to leptin, which suggests that new efferent signals emanate from the CNS after leptin treatment.     

Cadmium toxicity and distribution in metallothionein-I and -II deficient transgenic mice

To date, numerous correlative studies have implicated metallothionein in the detoxification of heavy metals and in the regulation of metal distribution within an organism. In the present study cadmium-binding proteins (metallothionein equivalents), cadmium acute toxicity, and cadmium distribution in tissues and subcellular fractions were compared in metallothionein-I and -II deficient (MT-/-) mice and the parental strain carrying intact metallothionein genes (MT+/+) to determine if the absence of metallothionein altered any of these parameters. In an uninduced state, MT-/- mice expressed lower levels of cadmium-binding proteins relative to MT+/+ mice in several tissues. Administration of zinc enhanced the levels of cadmium-binding proteins in liver, small intestine, kidney, pancreas, and male sex organs, but not in cecum or brain of MT+/+ mice compared to zinc pretreated MT-/- mice. The cadmium LD50 was similar for MT-/-, MT+/+, and zinc-pretreated MT-/- mice (15-17 mumol CdCl2/kg body weight delivered i.p.). However, zinc-pretreated MT+/+ mice had a cadmium LD50 of 58-63 mumol CdCl2/kg body weight. Over two-thirds of cadmium was found in liver, cecum, small intestine, and kidney in both MT+/+ and MT-/- mice; therefore, metallothionein levels do not appear to play a major role in the tissue distribution of cadmium. However, after zinc pretreatment, MT+/+ mice accumulated more cadmium in the liver and less in other tissues, whereas the amount of cadmium in the liver was not altered by zinc pretreatment in MT-/- mice. In general, the cytosolic/particulate ratio of cadmium was significantly higher in tissues of noninduced MT+/+ mice relative to MT-/- mice. This difference was accentuated after zinc pretreatment. Together these results indicate that basal levels of metallothionein do not protect from the acute toxicity of a single i.p. cadmium challenge. Furthermore, it does not appear that the cytosolic compartmentalization of cadmium is correlated with reduced toxicity.     

Alterations in lipoprotein metabolism in peroxisome proliferator-activated receptor alpha-deficient mice

The peroxisome proliferator-activated receptor-alpha (PPARalpha) controls gene expression in response to a diverse class of compounds collectively referred to as peroxisome proliferators. Whereas most known peroxisome proliferators are of exogenous origin and include hypolipidemic drugs and other industrial chemicals, several endogenous PPARalpha activators have been identified such as fatty acids and steroids. The latter finding and the fact that PPARalpha modulates target genes encoding enzymes involved in lipid metabolism suggest a role for PPARalpha in lipid metabolism. This was investigated in the PPARalpha-deficient mouse model. Basal levels of total serum cholesterol, high density lipoprotein cholesterol, hepatic apolipoprotein A-I mRNA, and serum apolipoprotein A-I in PPARalpha-deficient mice are significantly higher compared with wild-type controls. Treatment with the fibrate Wy 14,643 decreased apoA-I serum levels and hepatic mRNA levels in wild-type mice, whereas no effect was detected in the PPARalpha-deficient mice. Administration of the fibrate Wy 14,643 to wild-type mice results in marked depression of hepatic apolipoprotein C-III mRNA and serum triglycerides compared with untreated controls. In contrast, PPARalpha-deficient mice were unaffected by Wy 14,643 treatment. These studies demonstrate that PPARalpha modulates basal levels of serum cholesterol, in particular high density lipoprotein cholesterol, and establish that fibrate-induced modulation in hepatic apolipoprotein A-I, C-III mRNA, and serum triglycerides observed in wild-type mice is mediated by PPARalpha.     

Toxicity and metabolism in mice of 2,6-dithiopurine, a potential chemopreventive agent

2,6-Dithiopurine (DTP) has been proposed as a possible chemopreventive agent because of its facile reaction with the electrophilic ultimate carcinogen, benzo[a]pyrene diol epoxide, and other reactive electrophiles. Previous studies in mouse skin indicated almost complete inhibition of benzo[a]pyrene diol epoxide-induced tumorigenesis by DTP, suggesting the possible utility of this compound as a chemopreventive agent. However, little is known of the metabolism of DTP or of its possible long-term toxicity. Mice were fed diets containing up to 4% DTP in AIN-76A for a period of 7 weeks, and possible toxicity was monitored by weight gain and histopathological examination of all major tissues. No toxicity was observed at any dose of DTP. DTP was found to be a good substrate in vitro for two enzymes known to metabolize 6-mercapto-purine: xanthine oxidase and thiopurine methyltransferase. The in vitro metabolites were 2,6-dithiouric acid and an apparent monomethylated derivative, respectively. In vivo, the major urinary metabolite was 2,6-dithiouric acid, which attained levels as high as 34 mM in the urine of mice receiving the 4% DTP diet. DTP was also excreted unchanged in the feces and urine. DTP, 2,6-dithiouric acid, and an unidentified, relatively nonpolar metabolite were also detected in the serum of experimental animals. Although large interindividual variation in the serum DTP concentration was found, there was a dose-dependent increase in serum DTP as the dietary level of DTP was increased. These results suggest that neither toxicity nor metabolism will severely limit the utility of DTP as a chemopreventive agent.     

Kynurenic acid distribution into brain and peripheral tissues of mice

Kynurenic acid (KYN), an antagonist of excitatory amino acid receptors, is a putative antidote against neuroexcitatory amino acid toxicity. We studied various doses (0.05-3.17 mmol/kg, i.p.) and the effects of probenecid coadministration (0.70 mmol/kg, i.p.) on tissue distribution of KYN in male and female Swiss-Webster mice. After injection of [3H]KYN, samples of brain, heart, liver, kidney, skeletal muscle, and gut were collected at selected times and assayed for KYN by liquid scintillation counting. The substance was absorbed rapidly and distributed into all tissues. Its content (nmol/g, mean +/- SE) at 60 min was 0.26 +/- 0.05, 1.80 +/- 0.05, and 40.4 +/- 8.1 in brain (for 0.05, 0.53, and 3.17 mmol/kg), 1.43 +/- 0.11, 14.3 +/- 3.7, and 212 +/- 32 in heart, 1.16 +/- 0.21, 10.6 +/- 2.6, and 254 +/- 21 in liver, and 7.41 +/- 2.65, 180 +/- 63, and 1899 +/- 254 in kidney. Net accumulation of KYN in brain was much lower than in other tissues. Probenecid increased KYN concentration in brain 2.5-fold. Peak brain:blood concentration ratio occurred between 60 and 180 min, was inversely associated with dose, and was not affected by probenecid. Although brain content was similar, female mice had an earlier peak brain:blood ratio (120 min) than males (180 min) for the 0.05 mmol/kg dose. Our results suggest the presence of a restricted transfer process for KYN with delayed egress from brain.     

Effects of hormones on protein and amino acid metabolism in mammary-gland explants of mice

The effects of insulin, cortisol and prolactin on amino acid uptake and protein biosynthesis were determined in mammary-gland explants from mid-pregnant mice. Insulin stimulated [3H]leucine incorporation into protein within 15 min of adding insulin to the incubation medium. Insulin also had a rapid stimulatory effect on the rate of aminoiso[14C]butyric acid uptake, but it had no effect on the intracellular accumulation of [3H]leucine. Cortisol inhibited the rate of [3H]leucine incorporation into protein during the initial 4h of incubation, but it had no effect at subsequent times. [3H]Leucine uptake was unaffected by cortisol, but amino[14C]isobutyric acid uptake was inhibited after a 4h exposure period to this hormone. Prolactin stimulated the rate of [3H]leucine incorporation into protein when tissues were exposed to this hormone for 4h or more; up to 4h, however, no effect of prolactin was detected. At all times tested, prolactin had no effect on the uptake of either amino[14C]isobutyric acid or [3H]leucine. Incubation with actinomycin D abolished the prolactin stimulation of protein biosynthesis, but this antibiotic did not affect the insulin response. A distinct difference in the mechanism of action of these hormones on protein biosynthesis in the mammary gland is thus apparent.     

Effect of high dietary zinc upon zinc metabolism and intracellular distribution in cows and calves

Changes in the metabolism of zinc were investigated in mature nonlactating Holstein cows and 4-mo old calves fed high but nontoxic amounts of dietary zinc. Increasing dietary zinc of calves from 42 to 642 ppm elevated the zinc content of liver and pancreas 600% and 1400%, respectively. The elevated zinc in liver and pancreas involved a substantial increase in all intracellular fractions, with by far the largest amount associated with a 10,000 molecular-weight protein in the soluble cell fraction. This zinc-binding protein may protect the cell against damage from increased zinc. A comparable increase in dietary zinc of mature cows did not affect zinc content of liver or kidney nor alter the intracellular distribution of zinc in the liver. In mature cows, homeostatic control mechanisms which regulate the zinc content of tissue are much more effective than in calves. Accordingly, mature cows probably are able to tolerate higher concentrations of dietary zinc.     

Tissue distribution, metabolism, and clearance of the convulsant trimethylolpropane phosphate in rats

The distribution, metabolism, and clearance of trimethylolpropane phosphate (TMPP), a potent, bicyclophosphate, gamma-aminobutyric acid-ergic convulsant, were studied in male Fischer-344 rats. Intraperitoneal administration of TMPP was compared with oral gavage with respect to rates of absorption, distribution, and clearance. Distribution of TMPP to major body tissues was evaluated for the first 24 hr after administration or, in the case of regional brain distribution, immediately after the first TMPP-induced clinical seizure. Samples purified from the urine, feces, and bile of rats exposed to TMPP, as well as from rat liver microsomes incubated with TMPP in vitro, were analyzed for possible phase I and phase II metabolism, using HPLC. The disposition and clearance of TMPP in the blood and major body tissues were measured. TMPP was found to be well distributed to highly vascularized tissue compartments, with little retention >24 hr after administration. TMPP was eliminated through the urine and feces as the parent compound, with no evidence of phase I or phase II metabolism. TMPP was rapidly cleared from the blood during the first 30 min after exposure, with slower clearance of >87% of the drug during the following 8-hr period and >99.5% clearance by 100 hr after injection. Repeated daily exposure to TMPP for up to 5 successive days resulted in no measurable accumulation in the brain or other major tissue compartments. Possible mechanisms for TMPP-induced, short- and long-term, neurobehavioral modulation are discussed.