Increased retinoic acid metabolism following 3,3',4,4',5,5'-hexabromobiphenyl injection

Young male Wistar rats received single i.p. injections of 3,3',4,4',5,5'-hexabromobiphenyl. In rats dosed with 40 mg/kg, food consumption and growth as well as liver retinol and retinyl palmitate concentrations decreased, while serum retinol and liver weight increased within 28 days following the injection. In rats receiving a 20-mg/kg dose, food consumption, growth, liver weight, and serum retinol were not affect, although liver retinol and retinyl palmitate concentrations declined to 23 and 21% of their respective control values. Vitamin A metabolism was studied in liver microsomes prepared from rats sacrificed 7 days after the 20-mg/kg injection. The rate of retinoic acid hydroxylation via the cytochrome P-450 system to 4-hydroxyretinoic acid plus the subsequent oxidation to 4-ketoretinoic acid was significantly elevated. Retinoic acid conjugation by UDP-glucuronyl transferase was also significantly increased. These changes corresponded with increased activities of cytochrome P-450-dependent aryl hydrocarbon hydroxylase and UDP-glucuronyltransferase conjugation of p-nitrophenol. These results provide a direct link between enzyme induction due to xenobiotics and specific steps in the vitamin A metabolic pathway.     

Vitamin A metabolism in rats chronically treated with 3,3′,4,4′,5,′-hexabromobiphenyl

Chronic dietary administration of 3,3′,4,4′,5,5′-hexabromobiphenyl (HBB), 1 mg/kg diet, caused a decrease in retinol (20-fold) and retinyl esters (23-fold) in the livers of female rats, but resulted in a 6.4-fold increase in retinol and 7.4-fold increase in retinyl esters in the kidneys. Liver acyl-CoA: retinol acyltransferase and retinyl palmitate hydrolase activities were reduced while serum concentration of retinol was unaffected by HBB feeding. Metabolism of a physiological dose of [11-³H]retinyl acetate (10 μg), was examined in rats fed either vitamin A-adequate diet, or marginal amounts of vitamin A, or vitamin A-adequate diet containing HBB. A 13-fold greater amount of the administered vitamin A was found in kidneys of HBB-treated rats. In rats fed adequate or low amounts of vitamin A, kidney radioactivity was primarily in the retinol fraction, while in HBB-fed rats the radioactivity was associated mostly with retinyl esters. Fecal and urinary excretion of radioactivity was greatly increased in HBB-treated rats. Chronic HBB feeding results in a loss of ability of liver to store vitamin A, and severely alters the uptake and metabolism of vitamin A in the kidneys. We conclude that HBB causes major disturbances in the regulation of vitamin A metabolism.     

Increased thyroxine turnover after 3,3',4,4',5,5'-hexabromobiphenyl injection and lack of effect on peripheral triiodothyronine production

Juvenile male Wistar rats were injected i.p. with 0, 20, or 40 mg/kg 3,3',4,4',5,5'-hexabromobiphenyl and blood samples collected periodically up to 28 days. A dose-dependent depression of the serum thyroxine level was detected, while the circulating triiodothyronine concentration was not affected by the biphenyl congener. Thyroxine turnover in vivo 7 days after injection of the 20 mg/kg dose revealed significant increases of various clearance parameters relative to controls. The fractional clearance rate (day-1) increased by 84%, the daily metabolic clearance rate (mL.kg-1.day-1) increased by 128%, and the daily thyroxine disposal rate (ng.kg-1.day-1) increased by 41%. Also, the thyroxine distribution space (mL/kg) increased by 21%. These results indicated greater thyroxine binding in major organs as well as a marked increase in the peripheral metabolism of thyroxine. The increased thyroxine metabolism is explained by a 4.8-fold induction of uridine 5'-diphosphoglucuronyltransferase activity in liver microsomes. The type I 5'-deiodinase activity in liver homogenates and endogenous concentrations of the cofactor for this reaction, glutathione, were not affected by the biphenyl. This result means that homeostatic mechanisms involving thyroxine conversion to triiodothyronine do not explain the maintenance of serum T3 under these conditions.     

Degradation of 4,4'-dichlorobiphenyl, 3,3',4,4'-tetrachlorobiphenyl, and 2,2',4,4',5,5'-hexachlorobiphenyl by the white rot fungus Phanerochaete chrysosporium.

The white rot fungus Phanerochaete chrysosporium has demonstrated abilities to degrade many xenobiotic chemicals. In this study, the degradation of three model polychlorinated biphenyl (PCB) congeners (4,4'-dichlorobiphenyl [DCB], 3,3',4,4'-tetrachlorobiphenyl, and 2,2',4,4',5,5'-hexachlorobiphenyl) by P. chrysosporium in liquid culture was examined. After 28 days of incubation, 14C partitioning analysis indicated extensive degradation of DCB, including 11% mineralization. In contrast, there was negligible mineralization of the tetrachloro- or hexachlorobiphenyl and little evidence for any significant metabolism. With all of the model PCBs, a large fraction of the 14C was determined to be biomass bound. Results from a time course study done with 4,4'-[14C]DCB to examine 14C partitioning dynamics indicated that the biomass-bound 14C was likely attributable to nonspecific adsorption of the PCBs to the fungal hyphae. In a subsequent isotope trapping experiment, 4-chlorobenzoic acid and 4-chlorobenzyl alcohol were identified as metabolites produced from 4,4'-[14C]DCB. To the best of our knowledge, this the first report describing intermediates formed by P. chrysosporium during PCB degradation. Results from these experiments suggested similarities between P. chrysosporium and bacterial systems in terms of effects of congener chlorination degree and pattern on PCB metabolism and intermediates characteristic of the PCB degradation process.     

Effect of dietary selenium on the promotion of hepatocarcinogenesis by 3,3', 4,4'-tetrachlorobiphenyl and 2,2', 4,4', 5,5'-hexachlorobiphenyl

Polychlorinated biphenyls (PCBs) are persistent organic pollutants that have promoting activity in the liver. PCBs induce oxidative stress, which may influence carcinogenesis. Epidemiological studies strongly suggest an inverse relationship between dietary selenium (Se) and cancer. Despite evidence linking Se deficiency to hepatocellular carcinoma and liver necrosis, the underlying mechanisms for Se cancer protection in the liver remain to be determined. We examined the effect of dietary Se on the tumor promoting activities of two PCBs congeners, 3,3', 4,4'-tetrachlorobiphenyl (PCB-77) and 2,2', 4,4', 5,5'-hexachlorobiphenyl (PCB-153) using a 2-stage carcinogenesis model. An AIN-93 torula yeast-based purified diet containing 0.02 (deficient), 0.2 (adequate), or 2.0 mg (supplemental) selenium/kg diet was fed to Sprague-Dawley female rats starting ten days after administering a single dose of diethylnitrosamine (150 mg/kg). After being fed the selenium diets for 3 weeks, rats received four i.p. injections of either PCB-77 or PCB-153 (150 micromol/kg) administered every 14 days. The number of placental glutathione S-transferase (PGST)-positive foci per cm(3) and per liver among the PCB-77-treated rats was increased as the Se dietary level increased. Unlike PCB-77, rats receiving PCB-153 did not show the same Se dose-response effect; nevertheless, Se supplementation did not confer protection against foci development. However, the 2.0 ppm Se diet reduced the mean focal volume, indicating a possible protective effect by inhibiting progression of preneoplastic lesions into larger foci. Cell proliferation was not inhibited by Se in the liver of the PCB-treated groups. Se did not prevent the PCB-77-induced decrease of hepatic Se and associated reduction in glutathione peroxidase (GPx) activity. In contrast, thioredoxin reductase (TrxR) activity was not affected by the PCBs treatment or by Se supplementation. These findings indicate that Se does not inhibit the number of PGST-positive foci induced during promotion by PCBs, but that the size of the lesions may be inhibited. The effects of Se on altered hepatic foci do not correlate with its effects on GPx and TrxR.     

Antitumor effects of 3,3',4,4',5,5'-hexahydroxystilbene in HL-60 human promyelocytic leukemia cells

Resveratrol (3,4',5-trihydroxystilbene, RV) exerts remarkable cytostatic and cytotoxic effects against a multitude of human cancer cell lines. Since the introduction of additional hydroxyl groups was supposed to increase the biological activity of RV, we have synthesized a number of polyhydroxylated stilbene analogues as potential antitumor agents. In this study, the activity of 3,3',4,4',5,5'-hexahydroxystilbene (M8) was investigated in HL-60 human promyelocytic leukemia cells. Employing a growth inhibition assay, incubation with M8 and RV resulted in IC50 values of 6.25 and 12 microM, respectively. Using a specific Hoechst/propidium iodide double staining method, we found that M8 was able to induce apoptosis in concentrations significantly lower than those of RV. In addition, M8 arrested cells in the S phase and totally depleted cells in the G2-M phase of the cell cycle (143% and 0% of control after treatment with 12.5 microM M8, respectively). We therefore believe that this promising agent deserves further preclinical and in vivo testing.     

Fatty acid metabolism in neonatal chickens (Gallus domesticus) treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or 3,3',4,4',5-pentachlorobiphenyl (PCB-126) in ovo

Treatment of chickens as pre-incubation embryos with TCDD or PCB-126 altered fatty acid concentrations in their plasma 21 days later, compared with their oil vehicles (sunflower and corn oils, respectively). TCDD increased the concentrations of total fatty acids, lipid classes (phospholipids and cholesterol ester), fatty acid families (saturated, n-7 and n-6), and many specific fatty acids. The only fatty acid concentrations decreased by TCDD treatment were those of cholesterol ester fatty acids 20:3n3 and 24:6n3 and overall plasma 24:6n3. In contrast, PCB-126 treatment decreased total phospholipid, saturated and plasmogen fatty acid concentrations with generally decreasing trends in specific fatty acid concentrations. However, both TCDD and PCB-126 treatments increased total 22:1n9 and decreased 24:6n3 concentrations compared with their respective vehicles. The potential relationship between those fatty acid concentrations altered by toxicant treatment and alterations in brain symmetry was then examined using correlation analysis. Several fatty acid concentrations were significantly correlated with differences in brain morphology between the right and left hemispheres and these potential associations were different between toxicant and vehicle.     

Reduction of methemoglobin with photoradicals of benzophenone-3,3',4,4'-tetracarbonic acid

Reaction ability of partially reduced human methemoglobin was estimated by the rate of protein addition of carbon monoxide immediately after reduction. Impulse of photoexcitation allowed two variants of these experiments to be carried out: immediately after partial reduction of methemoglobin with photoradicals of benzophenone 3,3',4,4'-tetracarbon acid and after photodissociation of the carboxy-complex obtained in the first case. It has been shown that at protein reduction "p" less than or equal to 0.03 under these conditions the initially obtained R-form of protein is partially transformed into the less reactive T-form.     

Structural basis of species differences between human and experimental animal CYP1A1s in metabolism of 3,3',4,4',5-pentachlorobiphenyl

Coplanar polychlorinated biphenyls included in dioxin-like compounds are bio-accumulated and adversely affect wildlife and human health. Although many researchers have studied the metabolism of PCBs, there have been few reports of the in vitro metabolism of 3,3',4,4',5-pentachlorobiphenyl (PCB126), despite the fact that it has the highest toxicity among PCB congeners. Cytochrome P450 (CYP) 1A1 proteins can metabolize some dioxins and PCBs by hydroxylation, but the activities of human and rat CYP1A1 proteins are very different. The mechanism remains unclear. From our results, rat CYP1A1 metabolized PCB126 into 4-OH-3,3',4',5-tetrachlorobiphenyl and 4-OH-3,3',4',5,5'-pentachlorobiphenyl, but human CYP1A1 did not metabolize. Homology models of the two CYP proteins, and docking studies, showed that differences in the amino acid residues forming their substrate-binding cavities led to differences in the size and shape of the cavities; only the cavity of rat CYP1A1 allowed PCB126 close enough to the haem to be metabolized. Comparison of the amino acid residues of other mammalian CYP1A1 proteins suggested that rats have a unique metabolism of xenobiotics. Our results suggest that it is necessary to be careful in human extrapolation of toxicity data estimated by using the rat as an experimental animal, especially in the case of compounds metabolized by CYP1A1.     

3,3',4,4'-tetrachlorobiphenyl: metabolism by the chick embryo in ovo and toxicity of hydroxylated metabolites

The metabolism of 3,3',4,4'-tetrachlorobiphenyl (TCB) has been studied in the chicken in ovo by analysis of bile from chick embryos. Four percent of the [14C]TCB dose injected into the air sac on day 13 of incubation was detected in the bile by day 19. An increase of more lipophilic TCB metabolites was observed by HPLC analysis after hydrolysis of the bile. TCB and three phenolic TCB metabolites were identified and quantified in the hydrolyzed bile: TCB (14 ng/gall bladder), 5-hydroxy-3,3',4,4'-tetrachlorobiphenyl (234 ng/gall bladder), 4-hydroxy-3,3',4',5-tetrachlorobiphenyl (45 ng/gall bladder) and 2-hydroxy-3,3',4,4'-tetrachlorobiphenyl (3 ng/gall bladder). The presence of two other TCB metabolites in the bile, a dihydroxy-tetrachlorobiphenyl and a dihydroxy-trichlorobiphenyl was also indicated. The method used in the present study is well suited for studies of metabolism in avian embryos in ovo. The three TCB metabolites identified all proved to be at least two orders of magnitude less toxic than TCB in a chick embryo test. These metabolites were also shown to bind with significantly lower affinity than TCB to the Ah receptor. TCB, 5-hydroxy-3,3',4,4'-tetrachlorobiphenyl, 4-hydroxy-3,3',4',5-tetrachlorobiphenyl and 2-hydroxy-3,3',4,4'-tetrachlorobiphenyl gave Kd values of 16, 33, 45 and 37 nM, respectively, in the Ah receptor test.     

Comparison of particulate 3,3',5,5'-tetramethylbenzidine and 3,3'-diaminobenzidine as chromogenic substrates for immunoblot

In horseradish peroxidase (EC: 1.11.1.7)-dependent immunoblot assays, particulate 3,3',5,5'-tetramethylbenzidine (TMB) is shown to be a more efficient immunoblot substrate than the standard substrate 3,3'-diaminobenzidine (DAB), because TMB is easily prepared, stable, and less carcinogenic than is DAB. Assays of antibody in a serially diluted human immunodeficiency virus (HIV) control serum (CDC reference CAT# VS2151) have the same sensitivity limits with both DAB and TMB (1:312,500). Complete, working substrate solutions of H2O2/TMB/enhancer and of H2O2/DAB were stored at room temperatures and at 48 degrees C respectively. Periodic tests showed the TMB substrate system to be functional after four weeks at 48 degrees C and after eight weeks at room temperature, while the DAB system was functional after one week at 48 degrees C and after four weeks at room temperature. The stability, safety, and convenience of the commercially available TMB kits make this substrate ideal for immunoblot tests.     

Purification and characterization of the dog hepatic cytochrome P-450 isozyme responsible for the metabolism of 2,2',4,4',5,5'-hexachlorobiphenyl

The biochemical basis for the marked difference in the rate of the hepatic metabolism of 2,2',4,4',5,5'-hexachlorobiphenyl (245-HCB) by Beagle dogs and Sprague-Dawley rats has been investigated. Control dog liver microsomes metabolize this substrate 15 times faster than control rat liver microsomes. Upon treatment with phenobarbital (PB), at least two cytochrome P-450 isozymes are induced in the dog, and the hepatic microsomal metabolism of 245-HCB is increased on both a per nanomole P-450 basis (twofold) and a per milligram protein basis (fivefold). One of the PB-induced isozymes, PBD-2, has been purified to a specific content of 17-19 nmol/mg protein and to less than 95% homogeneity, as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In a reconstituted system containing cytochrome b5, this isozyme shows an activity toward 245-HCB which is greater than threefold that seen in intact liver microsomes from PB-induced dogs. A reconstituted system containing the major isozyme induced by PB in the rat (PB-B) metabolizes 245-HCB at 1/10 the rate observed with purified PBD-2. Antibody inhibition studies have shown that PBD-2 accounts for greater than 90% of the hepatic microsomal metabolism of 245-HCB in control and PB-induced dogs, while PB-B only accounts for about half of the metabolism of this compound by microsomes obtained from PB-treated rats. Immunoblot analysis has revealed that the level of PBD-2 in dog liver microsomes increases nearly sixfold with PB treatment, and this increase correlates well with the fivefold increase in the rate of hepatic microsomal metabolism of 245-HCB by dogs. Together these data support a primary role for isozyme PBD-2 in the hepatic metabolism of 245-HCB in control and PB-induced dogs. In addition, these results suggest that, in contrast to rats, dogs can readily metabolize 245-HCB as a result of the presence of a cytochrome P-450 isozyme with efficient 245-HCB metabolizing activity.     

Activation of peroxidase-catalyzed oxidation of 3,3',5,5'-tetramethylbenzidine with poly(salicylic acid 5-aminodisulfide)

5-Aminosalicylic acid (5-ASA) inhibited by a mixed mechanism the peroxidase catalyzed oxidation of tetramethylbenzidine (TMB) in 0.015 M phosphate-citrate buffer (pH 6.4) supplemented with 5% DMSO and 5% DMF. Poly(salicylic acid 5-aminodisulfide) (poly(SAADS)) in 0.01 M phosphate buffer (pH 6.2-7.4) supplemented with 5% DMSO and 5% DMF effectively activated the peroxidase-catalyzed oxidation of TMB. The activation was quantitatively characterized by coefficients (M^–1) determined at different pH values: increased linearly with increase in pH up to the maximal value of 2.44·10⁵ M^–1 at pH 7.0. The activating effect of poly(SAADS) on the peroxidase-catalyzed oxidation of TMB is explained by the activator properties of polyelectrolyte, with its anionic form interacting with peroxidase sites responsible for the acid-base catalysis.