Formulation effect on the dermal bioavailability of isothiazolone biocide.

A central tenet of the science of toxicology is that the toxic effect of any material monotonically increases with the amount of the material delivered to the target tissue. This is the so-called dose-response relationship. Previous work is discussed in which a dose-response relationship is established for the induction and elicitation of cutaneous contact allergy from the isothiazolone biocide which is a 3 to 1 mixture of 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one. The commercial product name for this mixture is Kathon LX biocide. Given a tissue dose-response, this current work determines and demonstrates a difference in risk from aqueous formulations with and without polymer. A lower delivered dose of biocide goes into the skin from an aqueous-based polymeric emulsion or paint than from an aqueous solution or oil-in-water emulsions without polymer. Thus, the estimate of risk, as a direct result of dose, and the determination of a safe use level of a biocide in any particular formulation matrix need to incorporate this reality.

Evaluation of auricular lymph node cell proliferation in isothiazolone-treated mice.

Chemicals that bind to protein may cause immunological responses that include allergic contact hypersensitivity mediated by T cells. Various animal models have been used to predict chemical-mediated contact sensitization. One assay that has recently been developed utilizes the proliferation response induced in the draining auricular lymph node after exposure to contact sensitizers. This assay has been modified to a simpler method that results in the more rapid processing of samples with greater reproducibility. Isothiazolones are chemicals used as biocides in various applications which at greater than use concentrations may cause allergic contact hypersensitivity. The mouse auricular lymph node cell proliferation assay has been used to examine the potential of various isothiazolones to induce contact sensitization. The extent of lymph node cell proliferation was dependent on the concentration of isothiazolone, the vehicle used in the application, and the ability of isothiazolone to bind to protein. Kathon biocide contains two isothiazolones: 5-chloro-2-methyl-4-isothiazolin-3-one (CMI)2 and 2-methyl-4-isothiazolin-3-one (MI). In vivo studies have shown that protein binding correlates with auricular lymph node cell proliferation and an increase in auricular lymph node size. CMI, which binds to protein, induced an auricular lymph node cell proliferation response while MI, which poorly binds to protein, neither stimulated a proliferative response nor induced an increase in lymph node size at concentrations similar to CMI.(ABSTRACT TRUNCATED AT 250 WORDS)

The histopathological and biochemical response of the stomach of male F344/N rats following two weeks of oral dosing with ethyl acrylate.

Male F344/N rats were dosed with ethyl acrylate (EA) either by daily gavage or in the drinking water for 2 weeks. The gavage dose levels were 0, 2, 10, 20, 50, 100, and 200 mg/kg; the drinking water dose concentrations were 0, 200, 1,000, 2,000, and 4,000 ppm (corresponding to 0, 23, 99, 197, and 369 mg/kg/day, respectively). In those animals dosed by gavage, irritation of the forestomach increased in incidence and severity over the 20-200 mg/kg dose range. In those animals dosed with EA in the drinking water, a much lower incidence of forestomach irritation and less severe lesions were observed at corresponding dose levels. No lesions were observed in the glandular stomach from either of the 2 modes of oral administration. Following 2 weeks of gavage dosing with EA, the total non-protein sulfhydryl (NPSH) content of the forestomach and glandular stomach, and the NPSH concentration of the liver were determined 2-24 hr after the last gavage dose. Animals dosed at 200 mg/kg reached approximately 11% of the initial NPSH content in the forestomach at 6 hr after dosing. NPSH depletion of this magnitude has been associated with cytotoxicity of other tissues in other studies. By contrast, either the glandular stomach nor liver were depleted of NPSH to levels generally associated with toxicity. These observations are consistent with the conclusion that bolus dosing of EA induces severe depletion of critical cellular thiols in the forestomach with toxic consequences, but not in the glandular stomach or liver. Changing the mode of oral administration for EA to continued small doses in the drinking water allowed efficient detoxification and did not induce sulfhydryl depletion or comparable forestomach toxicity at the same daily body burden.

UDP-glucuronosyltransferase activity toward digitoxigenin-monodigitoxoside. Differences in activation and induction properties in rat and mouse liver.

Glucuronidation of digitoxigenin-monodigitoxoside (DT1), a metabolite of the cardiac glycoside digitoxin, is mediated by the microsomal isozymes, UDP-glucuronosyltransferase(s) (UDP-GT). The present studies examined the activation and induction properties of UDP-GT activity toward DT1 in hepatic microsomes of rats and mice. When compared to enzyme activity present in native (latent) microsomes of the rat (0.104 +/- 0.010 nmol/min/mg of protein), the activity toward digitoxigenin-monodigitoxoside in mouse native microsomes was 3.5-fold higher (0.379 + 0.44 mumol/min/mg of protein). After treatment with ionic (sodium cholate), zwitterionic [3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS)], or nonionic (Emulgen 911, Triton X-100) detergents, or with UDP-N-acetylglucosamine, enzyme activity in rat microsomes remained unchanged. In contrast, UDP-GT activity (DT1) in mouse liver microsomes treated with detergents or with the nucleotide was increased 2-3-fold above native enzyme activity. Pretreatment of rats with the microsomal enzyme inducers, 3-methylcholanthrene and phenobarbital, had no effect on this enzyme activity, whereas pretreatment with pregnenolone-16 alpha-carbonitrile (PCN) and dexamethasone (DEX) increased enzyme activity toward DT1 800 and 380%, respectively. These findings support the hypothesis that PCN and DEX induce a unique form of UDP-GT in the rat that selectively glucuronidates DT1. In marked contrast, the activity of this enzyme in mouse liver was not affected by pretreatment with any of the microsomal inducers, including PCN and DEX. In both rat and mouse, the P-450p-dependent N-ethylmorphine demethylase activity was increased 10-15-fold in PCN-pretreated animals.(ABSTRACT TRUNCATED AT 250 WORDS)

The inducibility and ontogeny of rat liver UDP-glucuronyltransferase toward furosemide.

Furosemide (F) conjugation with glucuronic acid is the main pathway of F metabolism in humans and experimental animals. In order to study rat liver microsomal UDP-glucuronyltransferase (UDP-GT) activity towards F we developed an in vitro assay in which the conjugation product, furosemide 1-0-acyl glucuronide (FG) was separated and quantitatively determined by reverse phase high pressure liquid chromatography. The optimal conditions of the reaction were established and the apparent Km for F and UDP-glucuronic acid (UDPGA) were 0.22 and 1.76 mM, respectively. Substrate inhibition of UDP-GT toward F occurred at F concentrations higher than 1.5 mM. Developmental changes in F glucuronidation were compared to the ontogeny of UDP GT activity toward two other acceptors, 1-naphthol and estrone that are known to have different patterns of maturation. F glucuronidation was 26% of adult activity at 18 days of gestation, reached 48% at birth and gradually increased to 250% of adult activity at 22 days of age. Glucuronidation of 1-naphthol and estrone attained 87% and 44% of adult activity at 22 days of gestation, 37% and 66% in six-day-old rats and 100% and 427% of adult activity in 22-day-old rats, respectively. The effect of 3-methylcholanthrene (3-MC), phenobarbital (PB) and pregnenolone-16 alpha-carbonitrile (PCN) on F UDP-GT was studied and compared to their effect on 1-naphthol and estrone glucuronidation. PB, 3-MC and PCN increased F-UDP-GT activity to 208%, 282% and 342% of vehicle-treated animals, respectively, while F pretreatment did not affect the conjugation of F. In comparison, 1-naphthol glucuronidation was preferentially induced by 3-MC (4.4-fold of control) while estrone glucuronidation was induced by PB and PCN (4.9- and 2.5-fold of control, respectively). These studies suggest that several forms of UDP-GT activities, which differ in their ontogeny and inducibility patterns, are involved in the glucuronidation of F in vitro.

Induction of digitoxigenin monodigitoxoside UDP-glucuronosyltransferase activity by glucocorticoids and other inducers of cytochrome P-450p in primary monolayer cultures of adult rat hepatocytes and in human liver.

We have recently proposed that glucocorticoids induce cytochrome P-450p, a liver microsomal hemoprotein originally isolated from rats treated with the antiglucocorticoid pregnenolone 16 alpha-carbonitrile (PCN), through a mechanism that involves a stereospecific recognition system clearly distinguishable from the classic glucocorticoid receptor (Schuetz, E. G., Wrighton, S. A., Barwick, J. L., and Guzelian, P. S. (1984) J. Biol. Chem. 259, 1999-2012). We now report that digitoxigenin monodigitoxoside UDP-glucuronosyltransferase (DIG UDP-glucuronosyltransferase), a liver microsomal enzyme activity induced by PCN in rats, is also inducible, as is P-450p, in primary monolayer cultures of adult rat hepatocytes. DIG UDP-glucuronosyltransferase activity closely resembled reported characteristics of induction of P-450p in its time course of induction, concentration-response relationships, exclusivity of induction by steroids with glucocorticoid properties, unusual rank order of potency of glucocorticoid agonists, unusually high ED50 for induction by glucocorticoids, enhanced induction rather than inhibition by anti-glucocorticoids in the presence of glucocorticoids, and finally, induction by nonsteroidal inducers of P-450p. DIG UDP-glucuronosyltransferase activity was also readily detected in human liver microsomes and was elevated in two patients who had received inducers of P-450p. We conclude that the liver enzymes controlled by the postulated PCN recognition system include not only P-450p but also one or more UDP-glucuronosyltransferases.

Effects of butylated hydroxyanisole on acetaminophen hepatotoxicity and glucuronidation in vivo.

The present study examined the effects of butylated hydroxyanisole (BHA) on acetaminophen-induced hepatotoxicity and metabolism in vivo with emphasis on possible changes in the glucuronidation pathway. Female Swiss-Webster mice received BHA in the diet (1% w/w) for 12 days (600 to 800 mg/kg/day). BHA prevented acetaminophen hepatotoxicity (600 mg/kg, ip), based on serum alanine and aspartate aminotransferase activities and histopathological examination. The rate of elimination of acetaminophen from blood was 10-fold higher in BHA-fed mice (clearance, 49 ml/min/kg) than in controls (4.4 ml/min/kg). In general, the urinary metabolite excretion patterns in control and BHA-treated mice were the same. However, the rates of acetaminophen conjugation via the sulfation, glucuronidation, and mercapturic acid pathways were enhanced with the rate of glucuronide formation, the major biotransformation pathway of acetaminophen, increased sevenfold in BHA-treated mice (0.041 min-1) compared to controls (0.006 min-1). BHA increased hepatic UDP-glucuronosyltransferase activity twofold, as well as hepatic UDP-glucuronic acid concentrations. In addition, after acetaminophen administration, UDP-glucuronic acid in BHA-treated mice was depleted to a lesser extent and returned to control values more rapidly than in untreated animals. BHA had a similar but less pronounced effect on hepatic glutathione levels. The findings indicate that the rate of acetaminophen glucuronidation is increased in vivo during BHA feeding to mice. This effect appears to play a role in the enhanced excretion of acetaminophen as well as protection against acetaminophen-induced hepatotoxicity.

Effects of cysteine pro-drugs on acetaminophen-induced hepatotoxicity.

The effects of three cysteine pro-drugs on the hepatotoxicity and biotransformation of acetaminophen were examined to evaluate the factors responsible for antidotal effectiveness. N-Acetyl-L-cysteine, L-2-oxothiazolidine-4-carboxylate or the L- or D-isomers of 2-methylthiazolidine-4-carboxylate were administered to male mice immediately after a hepatotoxic dosage of acetaminophen (5.0 mmol/kg). In general the antidotal efficacies and potencies of the L-cysteine pro-drugs were similar; 5.0 mmol/kg prevented hepatotoxicity whereas moderate and no protection were observed after 1.65 and 0.55 mmol/kg, respectively. In contrast, the D-isomer of 2-methylthiazolidine-4-carboxylate was ineffective at all dosages. Both L-2-oxothiazolidine-4-carboxylate and L-2-methylthiazolidine-4-carboxylate enhanced blood acetaminophen elimination (28-31% decrease in half-life) whereas N-acetyl-L-cysteine and D-2-methylthiazolidine-4-carboxylate did not. The L-cysteine pro-drugs increased the urinary excretion of the cysteine and mercapturic acid conjugates of acetaminophen (34-119%) but did not alter excretion of acetaminophen-glucuronide or acetaminophen-sulfate. The D-cysteine pro-drug did not affect the urinary excretion of the acetaminophen metabolites examined. Biochemical analyses of the phase II pathway co-substrates, i.e., UDP-glucuronic acid, adenosine 3'-phospho-5'-phosphosulfate and glutathione, were performed on liver samples from mice treated with pro-drugs and/or acetaminophen. The pro-drugs exhibited their greatest effect on hepatic glutathione concentrations. Treatment with L-cysteine pro-drugs decreased the extent of depletion and/or increased the rate of repletion of hepatic glutathione levels after acetaminophen administration.(ABSTRACT TRUNCATED AT 250 WORDS)

Depletion of hepatic UDP-glucuronic acid by drugs that are glucuronidated.

Salicylamide, clofibric acid, valproic acid and chloramphenicol are all known to be glucuronidated. The effects of these compounds on the hepatic concentration of UDP-glucuronic acid, the cosubstrate for glucuronidation, were studied in mice and found to lower hepatic UDP-glucuronic acid in a dose- and time-dependent fashion. Valproic acid, chloramphenicol, salicylamide and clofibric acid depleted hepatic UDP-glucuronic acid significantly at dosages as low as 0.5, 0.5, 0.75 and 4.0 mmol/kg, respectively. Hepatic UDP-glucuronic acid was decreased by 90% by valproic acid, 91% by chloramphenicol, 98% by salicylamide and 41% by clofibric acid (after dosages of 4, 2, 1 and 5 mmol/kg, respectively). UDP-glucuronic acid was depleted maximally by 15 after drug administration. Salicylamide also was used as a model compound to study the effect of drug loading on the hepatic concentrations of UDP-glucose and glycogen, precursors of UDP-glucuronic acid. It was found that, in addition to UDP-glucuronic acid, salicylamide (4 mmol/kg) also depleted UDP-glucose and glycogen by about 50%. These data suggest that large drug loads cause an increase flux through the glucuronic acid pathway and that hepatic UDP-glucuronic acid is consumed more rapidly than it is produced.

Glucuronidation and sulfation in rabbit kidney.

Mammalian kidneys contain a heterogeneous population of cell types that perform a variety of diverse functions. The present study was undertaken to determine the relative intrarenal distribution of UDP-glucuronosyltransferase and sulfotransferase activities. Female rabbit kidneys were dissected, and homogenates of cortex, outer stripe of the medulla and proximal tubule segments were prepared. Proximal tubule segments, derived primarily from the S2 region of the tubule, were isolated using purely mechanical methods. UDP-Glucuronosyltransferase and sulfotransferase activities directed toward 1-naphthol were highest in proximal tubules (2.86 +/- 0.13 nmol and 133 +/- 13.1 pmol product formed/min/mg protein, respectively) compared to outer stripe (45% and 64% of proximal tubule activity, respectively) and cortex (61% and 45%, respectively). Detergent increased 1-naphthol glucuronidation in homogenates of cortex and medulla, but depressed activity in proximal tubules. Subcellular fractionation of proximal tubule cells by isopycnic and rate density centrifugation revealed that UDP-glucuronosyltransferase activity distributed with the denser components of the endoplasmic reticulum and/or Golgi. Moreover, although the majority of sulfotransferase activity distributed as free (cytosolic) protein, sulfotransferase activity was also observed in fractions containing denser components of the endoplasmic reticulum and Golgi. Proximal tubule segments also exhibited the highest specific activity of UDP-glucose dehydrogenase, the enzyme involved in UDP-glucuronic acid synthesis (1.5-fold higher in tubules than in other regions.(ABSTRACT TRUNCATED AT 250 WORDS)

Furosemide 1-O-acyl glucuronide. In vitro biosynthesis and pH-dependent isomerization to beta-glucuronidase-resistant forms.

A biosynthetic acyl-type glucuronic acid conjugate of furosemide was isolated from in vitro incubation of pregnenolone-16 alpha-carbonitrile-induced rat liver microsomes containing UDP-glucuronyltransferase activity, furosemide, and UDP-glucuronic acid. Furosemide 1-O-acyl glucuronide (FG) was specifically hydrolyzed by beta-glucuronidase (BG) and was also labile to alkaline hydrolysis. FG concentration decreased at an apparent first order rate when incubated at 37 degrees C in buffer solution of pH values greater than 6.0 with only moderate hydrolysis of the conjugate at pH values less than 8.5. Formation of rearrangement forms of FG that were resistant to BG but labile to alkaline hydrolysis accounted for most of the disappearance of FG at this pH range. Radiochemical labeling of the conjugate with either 14C-furosemide or 14C-UDP-glucuronic acid was detected in the BG-resistant isomerization products of FG as they were separated by HPLC. The structure of FG and its isomerization products was further verified by negative ion thermospray liquid chromatography/mass spectrometry. The abundant (M - 1)-ion at mass 505, the aglycone fragment at m/z 329, and the characteristic sugar fragment ion of mass 175 were found in the spectra of FG and three additional isomers. An ion at m/z 221 was noted only in the case of the parent conjugate and thus may prove to be a characteristic ion for 1-O-acyl-linked glucuronides under negative ion thermospray. In vivo as well as in vitro rearrangement of FG to BG-resistant forms might affect the results of furosemide disposition studies which use BG hydrolysis to determine FG formation.

Role of sulfhydryls in the hepatotoxicity of organic and metallic compounds.

Endogenous sulfhydryl compounds serve a critical role in maintaining the function and viability of living systems. Glutathione (GSH) is the most abundant of these nonprotein thiols. During the past decade it has been demonstrated that sulfhydryls such as GSH also serve an important role in protecting vital nucleophilic sites in the liver from electrophilic attack by numerous classes of reactive chemicals. Organocompounds such as bromobenzene and acetaminophen which undergo microsomal metabolism yield reactive intermediates that are specifically inactivated by conjugation with sulfhydryls in the form of GSH. Thus, for organocompounds GSH is extremely important in protecting against toxic insults. More recently, other sulfhydryl compounds also have been found to serve a specific but as yet less defined role in protecting biological systems against chemically induced injury. Metals such as cadmium have a high affinity for sulfhydryls and the metal binding protein metallothionein binds cadmium with high affinity. The highly specific association of the metal with this sulfhydryl-enriched protein serves to effectively sequester the reactive cadmium ion. The central role of sulfhydryl equivalents in the detoxication of organo- and metallocompounds is similar; however, the mechanism by which this is achieved is fundamentally different.

Effect of thyroid hormones on liver microsomal enzyme induction in rats exposed to 2,3,7,8,-tetrachlorodibenzo-p-dioxin.

The effect of thyroidectomy and thyroid hormone replacement therapy on liver microsomal enzyme induction was studied in 2,3,7,8,-tetrachlorodibenzo-p-dioxin (TCDD)-treated rats (100 micrograms/kg). Treatment of non-thyroidectomized rats with TCDD had no effect on the concentration of liver microsomal cytochrome b5. In contrast, cytochrome b5 content was increased by TCDD treatment of thyroidectomized rats, regardless of replacement therapy with either T3 or T4. TCDD treatment increased the concentration of cytochrome P-450 (2-3-fold) and the activities of benzo[a]pyrene hydroxylase (4-7-fold), ethoxyresorufin O-de-ethylase (50-70-fold) and UDP-glucuronosyltransferase (5-7-fold) in non-thyroidectomized and thyroidectomized as well as thyroidectomized thyroid hormone treated rats; indicating the induction of these liver microsomal enzyme activities is independent of thyroid status. Because thyroid status alters the toxicity of TCDD but does not alter the ability of TCDD to induce microsomal enzymes, it appears that TCDD toxicity may not be directly related to microsomal enzyme induction.

Effects of butylated hydroxyanisole on hepatic glucuronidation capacity in mice.

The present study has examined biochemical mechanisms by which butylated hydroxyanisole (BHA) increases the glucuronidation of xenobiotics. Male and female Swiss Webster mice received BHA in the diet (1% w/w) for 10 days (600 to 800 mg/kg/day). Hepatic UDP-glucuronosyltransferase activities were increased toward specific substrates in native and detergent-activated microsomes. In general, BHA increased glucuronidation toward group 1 substrates (1-naphthol and 4-nitrophenol) 36 to 141% whereas no changes were found with a group 2 (chloramphenicol) or a group 3 substrate (digitoxigenin monodigitoxoside). Also, activities toward unclassified substrates (estrone, acetaminophen, and diethylstilbestrol) were increased (29 to 139%) by BHA. BHA treatment also increased hepatic UDP-glucuronic acid content (two- to threefold) by increasing UDP-glucose concentration (30 to 50%) and enhancing UDP-glucose dehydrogenase activity (300 to 400%). BHA had a more pronounced effect in female than male mice when data were expressed as activity or amount per liver because BHA treatment selectively increased the liver weight in female mice. In conclusion, BHA increases the capacity for glucuronidation in mice by elevating both UDP-glucuronosyltransferase activities and UDP-glucuronic acid concentration in liver.

A radiometric method for the measurement of adenosine 3'-phosphate 5'-phosphosulfate in rat and mouse liver.

A rapid, sensitive, and specific procedure for the measurement of adenosine 3'-phosphate 5'-phosphosulfate (PAPS) in rat and mouse liver was developed. The method measures the formation of 1-[1-14C]naphthyl sulfate from 1-[1-14C]naphthol and PAPS (limiting substrate) via a sulfotransferase-catalyzed reaction. Conjugated and unconjugated 1-[1-14C]naphthol are separated by a single chloroform extraction. The method was validated with regard to reaction components, time, tissue preparation technique, and amount of tissue. Further, PAPS, when added to rat and mouse liver (30-120 nmol/g liver) prior to tissue preparation, was quantitatively recovered (87-101%). The assay can detect 150 pmol of PAPS and, consequently, the sensitivity to measure PAPS in mg quantities of liver was obtained. PAPS levels in rat and mouse liver were determined to be 59 +/- 5 and 20 +/- 3 nmol/g liver, respectively. The ease and sensitivity of this method make it suitable for routine use in pharmacologic and toxicologic studies.

Increased UDP-glucuronosyltransferase activity and UDP-glucuronic acid concentration in the small intestine of butylated hydroxyanisole-treated mice.

Butylated hydroxyanisole (BHA) has been shown to decrease the toxicological and carcinogenic potential of a variety of chemicals. One possible mechanism for chemoprotection is that BHA increases intestinal UDP-glucuronosyltransferase activity and thereby enhances the elimination of the toxicants. Given that oral ingestion is a major route of xenobiotic exposure, we have investigated the actions of BHA on the glucuronidation capacity in the upper small intestine of female mice. Ingestion of high dosages of BHA (600-800 mg/kg/day) for 10 days produced a significant increase in in vitro microsomal acetaminophen glucuronidation but not in diethylstilbestrol conjugation. Furthermore, the concentration of UDP-glucuronic acid, the co-substrate required for glucuronidation reactions, was increased almost 2-fold in small intestine. Ingestion of BHA also increased UDP-glucose concentration and UDP-glucose dehydrogenase activities approximately 2-fold. These findings show that BHA ingestion increases intestinal glucuronidation capacity and suggest that BHA may enhance the intestinal first-pass biotransformation of xenobiotics.

Acetaminophen decreases adenosine 3'-phosphate 5'-phosphosulfate and uridine diphosphoglucuronic acid in rat liver.

The purpose of this investigation was to determine the effects of acute administration of acetaminophen on adenosine 3'-phosphate 5'-phosphosulfate (PAPS) and UDP-glucuronic acid concentrations in fed male rats. Acetaminophen produced a dosage-dependent decline in rat hepatic PAPS concentrations which was significant after dosages of 150, 300, or 600 mg/kg, ip. The time course of the decline in PAPS values after 600 mg acetaminophen/kg showed that PAPS concentrations reached a nadir 1 hr after dosing (40% of control values). Serum sulfate concentrations were also decreased by large dosages of acetaminophen (32 and 15% of control 2 hr after 150 and 600 mg/kg, respectively) and a significant, positive correlation between serum sulfate and hepatic PAPS concentrations was noted. In addition, hepatic cysteine and glutathione concentrations were lowered by high dosages of acetaminophen. Hepatic UDP-glucuronic acid concentrations were greatly decreased for 2 hr after a dosage of 600 mg acetaminophen/kg (15, 23, and 42% of control 0.5, 1.0, and 2.0 hr after dosing, respectively) whereas 150 mg/kg produced a less pronounced and more transient decrease. These findings demonstrate that both PAPS and UDP-glucuronic acid concentrations in liver are decreased after administration of acetaminophen and imply that capacity-limited sulfation and glucuronidation of acetaminophen are due to a decrease in co-substrate availability.

Glutathione peroxidase and reductase activities in the aging mouse.

Previous results from this laboratory demonstrated that glutathione concentrations decrease in aging mouse tissues. In this investigation glutathione (GSH) peroxidase and glutathione (GSSG) reductase activities were measured in tissues of standardized aging mice. Methods were validated for the quantitative determination of both enzymes in liver, kidney and heart tissues. GSH peroxidase activities were 27-53% lower in liver, kidney and heart of very old (36 months) mice compared to mature (10 months) mice (P less than 0.01). The same aging decreases were found with either hydrogen peroxide or cumene hydroperoxide as substrate. In a similar way GSSG reductase activities in liver and kidney were 25-28% lower in the old versus mature mice (P less than 0.01), but heart levels were unchanged. Further the lower GSSG reductase levels were unaffected by FAD supplementation in vitro. The changes in specific activity for both enzymes were not due to changes in organ weights and total protein contents, which were constant from 10 to 36 months of age. These decreases in GSH peroxidase and GSSG reductase do not account for the lower GSH levels in aging. Of special importance, however, is that these decreases indicate that detoxification via glutathione peroxidase and glutathione reductase could be impaired in senescence.

Glutathione levels during the mosquito life span with emphasis on senescence.

Glutathione levels were determined in mosquitoes of all ages of the life span. Specific analyses for reduced (GSH) and oxidized (GSSG) glutathione were used and validated to ensure minimal autoxidation of GSH and conversion of these forms. Indeed GSH accounted for greater than 97% of the total glutathione (GSH + GSSG) content in all samples. Marked changes occurred during the life span, and the highest levels of GSH and total glutathione were found during larval growth and metamorphosis (P less than 0.001). Thereafter the levels decreased in the early adult, plateaued in the mature, and decreased 46% in the old and very old mosquito (P less than 0.001). This aging-specific decrease was a general phenomenon, for it occurred in all body regions of both sexes. Starvation up to 3 days did not affect the GSH levels. The importance of these changes in glutathione is its relationship to the reducing and biosynthetic capacities of different life span stages. Of special interest is the senescence decrease which can lead to lower biosynthetic activity and also impaired detoxification capacity.