Surface measurements of global warming causing atmospheric constituents in Korea.

The expansion of the industrial economy and the increase of population in Northeast Asian countries have caused much interest in climate monitoring related to global warming. However, new techniques and better platforms for the measurement of global warming and regional databases are still old-fashioned and are not being developed sufficiently. With respect to this agenda, since 1993, at the request of the World Meteorological Organization (WMO), to monitor functions of global warming, the Korea Meteorological Administration (KMA) has set up a Global Atmospheric Watch (GAW) Station on the western coast of Korea (Anmyun-do) and has been actively monitoring global warming over Northeast Asia. In addition, atmospheric carbon dioxide (CO2) has been measured for a similar KMA global warming program at Kosan, Cheju Island since 1990. Aerosol and radiation have also been measured at both sites as well as in Seoul. The observations have been analyzed using diagnostics of climate change in Northeast Asia and also have been internationally compared. Results indicate that greenhouse gases are in good statistic agreement with the NOAA/Climate Monitoring and Diagnostics Laboratory (CMDL) long-term trends of monthly mean concentrations and seasonal cycles. Atmospheric particulate matter has also been analyzed for particular Asian types in terms of optical depth, number concentration and size distribution.

Oltipraz-induced amelioration of acetaminophen hepatotoxicity in hamsters. II. Competitive shunt in metabolism via glucuronidation.

Previously, we demonstrated that oltipraz [OTP: 5-(2-pyrazinyl)-4-methyl-1,2-dithiol-3-thione] prevented the hepatotoxicity of acetaminophen (AAP) in hamsters and that the observed protection was not related to increases in hepatic reduced glutathione (GSH) levels. These experiments were designed to elucidate the mechanism of OTP-induced protection with respect to an apparent non-GSH-dependent system. Marked differences in the relative amounts of hepatic GSH content depleted by AAP in control vs OTP-treated hamsters occurred. Urinary recoveries of AAP and metabolites indicated that more AAP-glucuronide was formed at the expense of other major metabolites (AAP-GSH, -N-acetylcysteine, and -sulfate) in OTP-treated hamsters, while plasma toxicokinetic modeling suggested a greater rate of AAP systemic clearance. An increased apparent formation rate constant for AAP glucuronidation (135%), in concert with significantly lower apparent formation rate constants for those metabolites which reflect the production of the reactive intermediate from AAP (glutathione and N-acetylcysteine), provide the rationale for this shift of metabolism. The biochemical basis for metabolic shunting is significantly elevated hepatic UDP-glucuronic acid content, an increased calculated UDP-glucuronic acid synthetic rate, and an increased liver microsomal UDP-glucuronyl transferase activity in OTP-treated animals. These changes in AAP conjugation were concomitant with decreased fractional clearance of AAP via bioactivation and less in vivo AAP covalent binding. These data support the hypothesis that OTP provides a protecting effect from AAP hepatotoxicity due to an augmented and predisposing glucuronidation capacity.

Oltipraz-induced amelioration of acetaminophen hepatotoxicity in hamsters. I. Lack of dependence on glutathione.

These studies were designed to test the hypothesis that oltipraz (OTP) provided protection against AAP intoxication in a sensitive species, the hamster; and further, to show that the sparing effect was related to the marked increase in hepatic reduced glutathione (GSH) levels. Dose-response and time-course experiments demonstrated that maximal increases in liver GSH occurred at 48 hr after an oltipraz dose of approximately 2.0 mmol/kg (po). Accompanying greater GSH levels were increased glutathione disulfide (GSSG) levels. Decreased indices of the oxidation state of glutathione and of hepatic pyridine nucleotides indicated a greater share of glutathione existed as GSH and that increased reducing equivalents were present, respectively. Additionally, glutathione disulfide reductase activity was greater in OTP-treated groups. Glutathione S-transferase activities were only marginally increased. OTP treatment did not elicit observable hepatotoxicity, whereas AAP (2.6 mmol/kg, ip) resulted in a reproducible model of liver damage. OTP-treated groups were protected from AAP-induced toxicity, as shown by decreased plasma appearance of liver enzymes and unremarkable histopathology. However, the degree of liver GSH depletion by AAP was fourfold greater in non-OTP treated groups compared to those which had received the dithiolthione. To test the importance of increased hepatic GSH, the biosynthesis of glutathione was interrupted. Buthionine sulfoximine (BSO) treatment decreased hepatic GSH, the biosynthesis of glutathione was interrupted. Buthionine sulfoximine (BSO) treatment decreased hepatic GSH content to 50% of control in hamsters which either had or had not received OTP. The groups receiving BSO and AAP incurred 83% lethality, while no lethality, unremarkable liver histopathology, and plasma enzyme levels consistent with control were found in the group receiving OTP, BSO, and AAP. Treatment with BSO only had no influence on hepatotoxicity parameters. These results indicate that the increased GSH levels in the OTP-treated hamster are coincidental to the sparing effect of OTP and are not central to the protection scheme in AAP-induced hepatotoxicity.

Protective effects of selenium on acetaminophen-induced hepatotoxicity in the rat.

Experiments were undertaken to examine the ability of selenium to protect against acetaminophen-induced hepatotoxicity and to examine possible mechanisms for this protective effect. Pretreatment of male, Sprague-Dawley rats with sodium selenite (12.5 mumol Se/kg, ip) 24 hr prior to acetaminophen administration produced a significant protection against the hepatotoxic effects of acetaminophen as assessed by a decrease in the plasma appearance of alanine aminotransferase and aspartate aminotransferase activities following acetaminophen. This was accompanied by an increase in the hepatic glutathione levels in selenium-treated animals and an inhibition in the decrease in hepatic glutathione content observed in animals receiving hepatotoxic doses of acetaminophen. Selenium pretreatment decreased the in vivo covalent binding of acetaminophen metabolites to hepatic protein, but did not alter hepatic microsomal cytochrome P-450 content or NADPH cytochrome c reductase activity, suggesting that selenium does not significantly alter the metabolism of acetaminophen to reactive electrophilic metabolites by the cytochrome P-450-dependent mixed-function oxidase enzyme system. Selenium produced an increase in the activity of gamma-glutamylcysteine synthetase which may account for the increased glutathione availability in selenium-treated animals and increased the activities of glutathione S-transferase and glucose-6-phosphate dehydrogenase. Examination of the urinary metabolite profile in selenium-treated animals revealed that the urinary excretion of acetaminophen and its metabolites was significantly increased over a 72-hr period. The increase occurred in the AAP-glucuronide metabolite while parent AAP and AAP-sulfate were actually decreased in selenium-treated rats. No change in recovery was observed in the AAP-glutathione or AAP-mercapturate urinary metabolites. While the glutathione conjugating system is enhanced by selenium treatment, amelioration of acetaminophen toxicity is most likely the result of enhanced glucuronidation which effectively diverts the amount of acetaminophen to be converted by the cytochrome P-450 system to the toxic metabolite.

Circadian variations in hepatic glutathione content, gamma-glutamylcysteine synthetase and gamma-glutamyl transferase activities in mice.

Experiments were conducted to determine if the circadian variations previously observed in hepatic reduced glutathione (GSH) concentrations also occurred in the associated enzymatic activities involved in the synthesis (gamma-glutamylcysteine synthetase) and degradation (gamma-glutamyl transferase) of glutathione using male CF-1 mice. All parameters were measured at four hour intervals over a 24-h period under normal (L: 0600-1800 h) and reversed (L: 1800-0600 h) lighting schedules. Circadian rhythms were found in each parameter under both lighting schedules. With GSH content, the rhythms' peak occurred at 0200 h and the nadir at 1400 h under the normal lighting schedule and was reversed (peak: 1800 h; nadir; 0600 h) under reversed lighting. The enzymatic activities also varied in a circadian manner with a phase shift in the peak and nadir occurring with a change in lighting schedule. Liver weight varied in a circadian manner under both lighting schedules with greatest weights occurring at the end of the dark phase. These data show that not only does GSH content vary in a circadian manner but that associated enzyme activities do as well. However, the hepatic enzyme activities did not correlate well with the GSH rhythm and, thus, do not provide a mechanistic rationale for the GSH rhythm.

Differential effects of dietary selenium on hepatic and renal glutathione-related enzymes and on hepatic microsomal drug metabolism in the rat.

These studies were undertaken to assess the effect of dietary selenium on glutathione-related enzyme activities in the liver and kidney and on hepatic drug metabolism. The intent was to study underlying mechanisms of selenium-induced beneficial effects in some models of hepatoxicity and carcinogenesis. Dietary selenium, as sodium selenite, was incorporated into a torula yeast basal diet (0.02 ppm selenium) and fed to male rats at supplementation levels of 0.0-5.0 ppm selenium for periods of three or six weeks. Additionally, a commercial cereal-based diet (CD, 0.05-0.08 ppm selenium) was compared to the experimentally defined diet (DD) supplemented with approximately the same amount of selenium. Liver and kidney glutathione peroxidase activity essentially plateaued at levels of selenium of 0.1 ppm and greater. CD- and DD-fed animals had hepatic and renal glutathione peroxidase activities which were similar. Glutathione S-transferase activity in liver, but not kidney, increased with increasing supplements of selenium. Glutathione S-transferase activities in CD- and DD-fed rats were not different. Cytochrome P-450 content and associated oxidative drug metabolism activities were relatively unmodified by selenium. Overall, dietary selenium appeared to act by enhancing potential conjugative detoxication pathway, rather than by decreasing the potential activation of chemicals via the hepatic cytochrome P-450 system.

Comparison of basal glutathione S-transferase activities and of the influence of phenobarbital, butylated hydroxy-anisole or 5,5'-diphenylhydantoin on enzyme activity in male rodents.

1. Hepatic cytosolic glutathione S-transferase (GST) activities, toward five substrates, were shown to vary markedly among three laboratory rodent species. 2. Basal GST activities for 1-chloro-2,4-dinitrobenzene (hamster greater than mouse greater than rat), 1,2-dichloro-4-nitrobenzene (mouse greater than rat greater than hamster), p-nitrobenzyl chloride (rat = mouse = hamster), bromosulfophthalein (rat greater than mouse greater than hamster) and 1,2-epoxy-3-(p-nitrophenoxy)propane (mouse greater than rat = hamster) differed with respect to magnitude and distribution among species. 3. GST substrate activities in response to phenobarbital, butylated hydroxy-anisole or 5,5'-diphenylhydantoin treatment were increased more often in mouse and rat as compared to the hamster. 4. These results suggest that basal GST activity, as well as inducibility, differ among rodent species. Since GST are involved in detoxication processes, differences in GST properties may underlie variability in species sensitivity to toxicants.

Effect of sodium selenite upon bromobenzene toxicity in rats. I. Hepatotoxicity.

The effects of sodium selenite on bromobenzene hepatotoxicity were examined in male rats. Rats pretreated with sodium selenite (12.5 or 30 mumol/kg, ip) 72 hr prior to injection of bromobenzene (7.5 mmol/kg, ip) showed a marked reduction in bromobenzene-induced liver injury as evidenced by decreased plasma alanine and aspartate transaminase values, sorbitol dehydrogenase activity, and reduced histologic damage. Administration of bromobenzene did not affect the selenium content of blood or liver. At 72 hr after treatment with selenite, hepatic reduced (GSH) and oxidized (GSSG) glutathione values or GSH synthetic and degradation enzyme activities were not altered. However, from 3 to 12 hr following bromobenzene administration, hepatic GSH and cysteine amounts declined less rapidly in selenite-treated rats compared to control. Thus, acute selenite treatment ameliorated bromobenzene hepatotoxicity in a manner suggesting facilitation of hepatic GSH production by selenite for use in bromobenzene detoxication.

Effect of sodium selenite upon bromobenzene toxicity in rats. II. Metabolism.

The effects of sodium selenite (12.5 or 30 mumol/kg, ip) upon bromobenzene metabolism were examined in male rats treated with selenite at 72 hr prior to bromobenzene exposure (7.5 mmol/kg, ip). The inhibitory nature of selenium treatment upon xenobiotic metabolism and increased production of hepatic thiol suggested that selenite might affect metabolic activation and detoxification of bromobenzene. Selenite treatment lowered in vivo covalent binding of [14C]bromobenzene while the in vitro covalent binding of [14C]bromobenzene in microsomes isolated from selenite-treated rats was unaffected compared to control. When the rate of [14C]bromobenzene decline in whole blood was evaluated in selenite-treated rats over 48 hr after bromobenzene administration, no significant differences were observed when compared to control. Furthermore, values of glutathione conjugates and hydroxylated metabolites of bromobenzene were similar in urine samples collected over 48 hr from selenite and control rats. Mechanistically, reduction of bromobenzene hepatotoxicity by selenite is not mediated through an altered metabolism of bromobenzene, but by alteration of cellular events occurring after metabolic activation.

Amelioration of bromobenzene hepatotoxicity in the male rat by zinc.

Experiments were conducted to examine the role of zinc in the prevention of bromobenzene hepatoxicity in male rats. Bromobenzene (BB) (7.5 mmol/kg, ip) produced a marked hepatotoxicity as evidenced by increases in plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities and a marked depression in hepatic glutathione (GSH) content 24 hr after administration. The administration of zinc (92 mumol Zn/kg, ip, at 48 and 24 hr prior to the bromobenzene) ameliorated the bromobenzene elevations in plasma AST (25%) and plasma ALT (50%) but did not alter the decreases in hepatic GSH. Following administration of [14C]BB, the radioactive label was distributed primarily in the cytosolic and lipid fractions derived from liver homogenates. Furthermore, the subcellular distribution of [14C]BB was not altered by zinc pretreatment. The extent of covalent binding of [14C]BB metabolites to hepatic tissue was significantly depressed in zinc-treated rats. Zinc induced the hepatic levels of metallothionein but [14C]BB did not bind to this sulfhydryl rich protein. Further experiments showed that zinc treatment depressed cytochrome P-450 content, the activity of NADPH cytochrome c reductase, and the metabolism of aniline, but not that of ethylmorphine. These studies suggest that the hepatoprotective effect of zinc against bromobenzene toxicity does not involve altered binding of the reactive toxic metabolite to glutathione or metallothionein, but it may be mediated by the inhibitory effect of zinc on the microsomal cytochrome P-450-dependent drug metabolizing system.

Alterations in hepatic microsomal drug metabolism and cytochrome P-450 proteins in spontaneously hypertensive rats.

Experiments were conducted to determine if substrate-specific changes in microsomal metabolism and liver proteins occurred in young (12-13 weeks) spontaneously hypertensive rats (SHR) fed ad libitum compared to age-matched normotensive Wistar Kyoto (WKY) control rats. The hepatic microsomal protein content in SHR rats was significantly increased compared to WKY rats while cytosolic and total liver protein levels did not differ between the two groups. Liver microsomal ethylmorphine-N-demethylase activity was substantially enhanced in SHR rats with only slight increases in cytochrome P-450 content and aniline hydroxylase activity compared to WKY rats. The substrate-specific increases in the microsomal drug metabolism in SHR rats were accompanied by an increase in the prominence of a protein with molecular weight 55,000 in the cytochrome P-450 region. These preliminary observations may be clinically relevant in that alterations in hepatic drug metabolism may be associated with endogenous biochemical processes underlying the hypertensive state.

Differential effects of dietary selenium on hepatic and renal glutathione metabolism in the rat.

Studies were undertaken to determine whether or not dietary selenium (Se), as sodium selenite, at suboptimal (unsupplemented torula yeast diets, 0.02 ppm Se) or supplemented up to dietary excess, but nontoxic, levels (5.0 ppm Se) could selectively modify hepatic and renal reduced glutathione (GSH) levels and the enzymes involved in GSH metabolism. Male rats were provided torula yeast semipurified diets containing approximately 0.02 ppm Se in the basal diet and supplements of 0.1, 2.0, or 5.0 ppm Se for 3 or 6 weeks. Hepatic GSH increased in a nonlinear manner, with increasing dietary Se at both time points. Renal GSH was not similarly influenced. Neither hepatic nor renal gamma-glutamylcysteine synthetase or gamma-glutamyl transpeptidase activities are altered by supplements of Se. This suggests that synthetic and degradation enzyme activities are not influenced by Se. The capacity for the maintenance of GSH in the reduced state by glutathione reductase activity increased with increasing levels of dietary Se in the liver but not in the kidney. In both tissues greater Se supplements yielded greater tissue burdens of Se. These results suggest that GSH metabolism in hepatic and renal systems is differentially mediated, and the basis for these differences could be influenced by the relative levels in glutathione metabolizing enzymes.

Effect of manganese on the hepatic microsomal mixed function oxidase enzyme system in the rat.

Experiments were conducted to examine the effect of manganese on the hepatic mixed function oxidase system in the rat. Acute treatment with manganese chloride (1-10 mg Mn/kg, ip) produced a significant prolongation of hexobarbital hypnosis in male rats on Days 2 and 3 following metal administration. The threshold dose of manganese to produce this alteration in response was 5 mg Mn/kg and the altered response returned to control values by Day 5. The prolonged hexobarbital hypnosis resulted from Mn inhibition of the hepatic microsomal mixed function oxidase system, the activity of which was assessed using aniline (23%), ethylmorphine (26%), and hexobarbital (27%) as substrates. Manganese treatment also produced significantly reduced levels of cytochrome P-450 (23%) and b5 (21%), but the substrate-induced spectral binding of all three substrates was not altered significantly by Mn when expressed as delta A per nanomole of cytochrome P-450. The activity of NADPH cytochrome c reductase was also significantly decreased (25%) by Mn treatment. Following the in vitro addition of Mn in concentrations ranging from 1 X 10(-6) to 1 X 10(-3) M Mn to microsomes derived from naive rats, there was no decrease in the metabolism of aniline or hexobarbital or cytochrome P-450 levels. Significant inhibition in ethylmorphine metabolism was observed with Mn concentrations of 1 X 10(-4) M and greater. These experiments indicate that acute Mn treatment can alter drug response as the result of decreased hepatic biotransformation which occurs by an indirect mechanism.

Bladder freeze ulceration and sodium saccharin feeding in the rat: examination for urinary nitrosamines, mutagens and bacteria, and effects on hepatic microsomal enzymes.

We previously demonstrated that long-term feeding of sodium saccharin, a non-mutagen, induced bladder carcinomas when administered to F344 male rats with regenerative hyperplasia of the urothelium induced by the freeze-ulceration technique, even without prior chemical initiation (Cohen et al. Cancer Res. 1982, 42, 65). In the present study, we examined the urine of rats subjected to freeze ulceration of the bladder and then fed sodium saccharin at 5% in the diet to evaluate the possibility of a mutagen being generated as a result of ulceration and/or saccharin feeding. Urine was collected into a syringe by aspiration from the urinary bladder after ligating the urethra for 2 hr at intervals from day 0 to day 14 after ulceration. After ulceration and/or sodium saccharin feeding, the urine showed no bacterial contamination, no mutagenic activity in the standard Ames assay, no production of nitrosamines, and no nitrosating environment. In addition, no significant changes in activities of liver microsomal enzymes (i.e. cytochrome P-450, NADPH-cytochrome c reductase, aniline hydroxylase, or ethylmorphine N-demethylase) were observed in rats fed sodium saccharin for 1, 5 or 14 days. Thus, freeze ulceration, and the consequent regenerative hyperplasia of the epithelium, compared with sodium saccharin feeding do not involve the administration of an exogenous mutagenic substance or the generation of a detectable mutagen in the urine.

Direct enzymatic assay for reduced and oxidized glutathione.

A modification of a specific assay for the reduced (GSH) and oxidized (GSSG) species of glutathione is presented and compared with the method of Ellman (1959). The present method has an enzymatic basis using GSH as the specific cosubstrate for glutathione S-transferase activity. The enzymatic method resulted in comparable, but consistently lower, values for GSH and GSSG than did the method of Ellman. The greatest differences between the two methods occurred when measuring renal GSH and GSSG, possibly due to the presence of mixed thiols in the kidney. This enzymatic method was more specific and provided an accurate and reproducible method of GSH and GSSG determination.

Effect of acute and repeated chlordimeform treatment on rat hepatic microsomal drug metabolizing enzymes.

The effect of chlordimeform treatment on the hepatic microsomal drug metabolizing enzymes was examined in male and female rats following either acute or repeated treatment. After acute administration of chlordimeform (100 mg/kg, ip one hr prior to sacrifice) differential effects were observed in various parameters of the hepatic microsomal mixed function oxidase system with significant decreases in ethylmorphine metabolism, cytochrome P-450 content, NADPH cytochrome c reductase, and in the spectral binding of hexobarbital and aniline while no changes were found in the metabolism of aniline or p-nitroanisole. Durations of zoxazolamine-induced paralysis and pentobarbital-induced hypnosis were increased significantly after acute chlordimeform administration. Following repeated administration of chlordimeform (75 mg/kg ip for four days) to adult male rats, a decrease was observed in zoxazolamine-induced paralysis time while pentobarbital-induced hypnosis was not altered. Metabolism studies using isolated hepatic microsomal fractions showed a decrease rate of biotransformation of ethylmorphine and aniline while the activity of p-nitroanisole O-demethylase was not changed. No differences were found in cytochrome P-450 levels whereas microsomal spectral binding of hexobarbital was reduced while that of aniline was not affected. Following acute or repeated administration of chlordimeform to adult female rats, decreases in the hepatic microsomal metabolism of aniline, but not ethylmorphine or p-nitroanisole, were observed. Addition of chlordimeform to microsomal suspensions yielded a Type I spectral binding curve.

Effect of manganese on hepatic drug metabolism in male and female rats.

Experiments were conducted to examine the effect of manganese on drug response and metabolism in male and female rats. Three days after administration of manganese chloride (5 mg Mn/kg, ip), the duration of hexobarbital hypnosis was prolonged in male but not in female rats. Hepatic microsomal metabolism of aniline, ethylmorphine, and hexobarbital was significantly inhibited in male rats. Metabolism of aniline and ethylmorphine was also inhibited in female rats but to a lesser extent than in males. Hexobarbital metabolism was not inhibited in female rats. Cytochrome P-450 content was depressed only in male rats, while cytochrome b5 levels were decreased in both sexes. These results indicate that sex-related differences exist in the ability of manganese to alter drug response and metabolism.