Increased toxicity of benzo(a)pyrene-7,8-dihydrodiol in the presence of polychlorobiphenylols

Benzo(a)pyrene (BaP) and polychlorinated biphenyls (PCBs) often co-exist in contaminated environments. Polychlorobiphenylols (OH-PCBs), formed by CYP-dependent monooxygenation of PCBs, are potent inhibitors of the glucuronidation of hydroxylated BaP metabolites. We hypothesized that OH-PCBs could drive the biotransformation of (-)BaP-7,8-dihydrodiol (BaP-7, 8-D) away from detoxication and towards formation of the reactive metabolite. A mixture of five OH-PCBs with 4-6 Cl atoms was infused into isolated, perfused, biliary intact livers (n=3 fish) removed from 3-methylcholanthrene-induced channel catfish. Controls (n=3) were infused with vehicle. Subsequently, [3H]-BaP-7, 8-D was infused into each liver and bile was collected for 1 h. The livers were taken for analysis of metabolites and DNA adducts. Induction status was confirmed by EROD assay. Bile was analyzed for metabolites. It was found that preinfusion of the mixture of OH-PCBs reduced the extent of glucuronidation of BaP-7, 8-D and increased the formation of DNA adducts 5-fold over controls. GSH conjugates, tetrols and triols were increased in the OH-PCB-infused fish, providing further support for our hypothesis that if the glucuronidation were inhibited, CYP-dependent activation would increase. These studies suggest a mechanism for synergy of toxicity of PAH and PCBs.     

Increased toxicity of benzo(a)pyrene-7,8-dihydrodiol in the presence of polychlorobiphenylols

Benzo(a)pyrene (BaP) and polychlorinated biphenyls (PCBs) often co-exist in contaminated environments. Polychlorobiphenylols (OH-PCBs), formed by CYP-dependent monooxygenation of PCBs, are potent inhibitors of the glucuronidation of hydroxylated BaP metabolites. We hypothesized that OH-PCBs could drive the biotransformation of (−)BaP-7,8-dihydrodiol (BaP-7, 8-D) away from detoxication and towards formation of the reactive metabolite. A mixture of five OH-PCBs with 4–6 Cl atoms was infused into isolated, perfused, biliary intact livers (n=3 fish) removed from 3-methylcholanthrene-induced channel catfish. Controls (n=3) were infused with vehicle. Subsequently, [³H]-BaP-7, 8-D was infused into each liver and bile was collected for 1 h. The livers were taken for analysis of metabolites and DNA adducts. Induction status was confirmed by EROD assay. Bile was analyzed for metabolites. It was found that preinfusion of the mixture of OH-PCBs reduced the extent of glucuronidation of BaP-7, 8-D and increased the formation of DNA adducts 5-fold over controls. GSH conjugates, tetrols and triols were increased in the OH-PCB-infused fish, providing further support for our hypothesis that if the glucuronidation were inhibited, CYP-dependent activation would increase. These studies suggest a mechanism for synergy of toxicity of PAH and PCBs.     

Glucuronidation in the polar bear (Ursus maritimus)

Polar bears bioaccumulate lipophilic pollutants, including polychlorinated biphenyls (PCBs), into their bodies from their exclusive diet of marine organisms. Hydroxylated PCB metabolites (OH-PCBs) have been found in plasma, presumably due to CYP-dependent biotransformation of PCBs in liver. Little is known about the phase 2 metabolism of hydroxylated xenobiotics in polar bears. The objective of this study was to examine UDP-glucuronosyltransferase (UGT) activity with OH-PCBs and a hydroxylated polycyclic aromatic hydrocarbon, 3-hydroxy-benzo(a)pyrene (3-OH-BaP), in polar bear liver. Samples of frozen polar bear liver were used to prepare microsomes. UGT activity with 3-OH-BaP in Brij-treated microsomes, measured by a fluorescence assay, was readily measurable with protein concentrations in assay tubes of up to 10 g/ml, but dropped off very sharply at higher protein concentrations. The apparent Km for 3-OH-BaP was 1.71 +/- 0.04 microM, and Vmax 1.26 +/- 0.16 nmol/min/mg protein (mean +/- SD, n=3). UGT activities with a model tetrachloro-OH-PCB (4'-OH-CB72) and a model hexachloro-OH-PCB (4'-OH-CB159) were assayed with [14-C]-UDPGA and separation of the [14-C]-glucuronide by ion-pair extraction and thin-layer chromatography. [14-C]-glucuronide conjugates were readily formed by polar bear liver microsomes in the absence of added substrate, apparently from contaminants present in liver. This phenomenon was not observed using hepatic microsomes from laboratory-held catfish. Glucuronidation efficiency was much higher with 4'-OH-CB72 (Km 7.3 microM; Vmax 1.55 nmol/min/mg) than 4'-OH-CB159 (Km 16.1 microM; Vmax 0.46 nmol/min/mg). The identities of the aglycones present in polar bear liver are not known, but could include OH-PCBs or hydroxylated metabolites of other persistent organic pollutants. This study demonstrates that UGT with high activity for 3-OH-BaP and other substrates is present in polar bear liver.     

Kinetic and toxicological characteristics of acetylcholinesterase from the gills of oysters (Crassostrea rhizophorae) and other aquatic species

The aim of this work was to characterize the cholinesterases from gills of Crassostrea rhizophorae in order to use them as biomarkers. Gills were homogenized and then centrifuged (9,000 x g, 4 degrees C, 30 min). S9 and Triton X-100 S9 treated (TX S9) fractions were employed as enzyme source. Km(ap) and Vmax were estimated, using acetylthiocholine iodide as substrate. Inhibition assays were performed with iso-OMPA and eserine. The Km(ap) for S9 and TX S9 fractions were 0.05 and 0.06 mM, whereas the Vmax were 1.92 and 5.84 nmol/min/mg protein. respectively. No inhibition was detected when the samples were incubated with iso-OMPA, suggesting the presence of acetylcholinesterases (AChE) in oyster gill homogenates. Sensitivity to eserine inhibition of AChE in the gills of oysters is intermediate when compared with other aquatic species.     

In-vitro metabolism of benzo[a]pyrene and benzo[a]pyrene-7,8-dihydrodiol by liver and intestinal mucosa homogenates from the winter flounder (Pseudopleuronectes americanus)

Freshly prepared homogenates were used to assess the relative ability of winter flounder (Pseudopleuronectes americanus) liver and intestinal mucosal cells to metabolize the polycyclic aromatic hydrocarbon (PAH) benzo[a]pyrene (BaP) and its proximate carcinogenic metabolite, BaP-7,8-dihydrodiol (7,8-Diol). Data obtained from homogenates prepared from fish previously fed β-naphthoflavone (BNF) indicated that both tissues had similar abilities to metabolize either BaP or 7,8-Diol on a per gram of protein basis. Metabolite profiles produced indicate that water-soluble metabolite formation is favored at low doses. These findings support the hypothesis that the intestine plays an important role in first-pass metabolism of dietary carcinogens in the winter flounder.     

Induction of phenol-type sulfotransferase and glucuronosyltransferase in channel catfish and mummichog

Conjugation of phenolic xenobiotics and metabolites through sulfation and glucuronidation is an important biotransformation pathway. Sulfotransferases (SULT) are generally considered non-inducible, while some UDP-glucuronosyltransferase (UGT) isoenzymes are co-induced with cytochrome P450-1A by Ah-receptor ligands. To test these assumptions for two fish species, we measured sulfation and glucuronidation of 9-hydroxybenzo[a]pyrene in 3-methylcholanthrene (3-MC) treated channel catfish (Ictalurus punctatus) and in mummichog (Fundulus heteroclitus) from the creosote contaminated Atlantic Wood site in the Elizabeth River, VA. The results show a significant induction of both UGT and SULT activity in 3-MC treated catfish, linked to the expected induction of EROD activity. In mummichog, significant induction of UGT was measured at the contaminated site over the reference site (King's Creek, VA), as well as extremely low SULT activities at both sites. Western blots, using a polyclonal antibody for catfish phenol-type SULT, confirmed the absence of phenol-type SULT in mummichog. Residual, though slightly inducible, SULT activity may be attributed to other SULT isoforms.     

Esterification of vertebrate-like steroids in the eastern oyster (Crassostrea virginica)

The esterification of two model vertebrate steroid hormones - estradiol (E2) and dehidroepiandrosterone (DHEA) - was studied in the oyster Crassostrea virginica. The activity of acyl-CoA:steroid acyltransferase was characterized in microsomal fractions isolated from oyster digestive glands. The apparent Km and Vmax values changed with the fatty acid acyl-CoA used (C20:4, C18:2, C18:1, C16:1, C18:0 or C16:0), and were in the range of 9-17 microM, and 35-74 pmol/min/mg protein for E2, and in the range of 45-120 microM, and 30-182 pmol/min/mg protein for DHEA. Kinetic parameters were also assessed in gonadal tissue. The enzyme saturated at similar concentrations, although conjugation rates were lower than in digestive gland. Preliminary data shows that tributyltin (TBT) in the low microM range (1-50) strongly inhibits E2 and DHEA esterification, the esterification of E2 being more sensitive to inhibition than that of DHEA. Overall, results indicate that apolar conjugation occurs in oysters, in both digestive gland and gonads, at a very similar rate to mammals, suggesting that this is a well conserved conjugation pathway during evolution. Esterification, together with other mechanisms, can modulate endogenous steroid levels in C. virginica, and might be a target for endocrine disrupters, such as TBT.     

Seasonal changes in phosphatase activities in Toulon Bay (France)

We studied the characteristics of the phosphatase activity (Km and Vmax) in total seawater and in particulate material of the three main plankton classes (0.25-5, 5-90 and >90 microm) in a coastal marine ecosystem of Toulon Bay (French Mediterranean Sea). The measurement of the hydrolysis of sodium paranitrophenylphosphate (pNPP), a substrate of phosphatase, revealed low and high affinity components in unfiltered seawater and in particulate matter. In unfiltered seawater, the low affinity activity was predominant from October to March during phytoplankton development. The high affinity activity dominated from April to June and was significantly correlated with the bacterial abundances. The phosphatase behaviour in the particulate material differs from that in the unfiltered seawater. The activity of the three particulate classes was generally much lower than that of unfiltered seawater, particularly the low affinity activity. The >90 microm size fraction consisted in greater part of zooplankton. In this size class, the activity (nmol l(-1) h(-1)) of the low affinity component was predominant from May to August, when the abundance of the larvae of copepods (copepodites) was highest. Its high specific activity (Activity/Protein concentration as nmol l(-1) h(-1) microg(-1)) was particularly elevated during this period. The 5-90 microm fraction consisted of phytoplankton cells, especially Dinoflagellates. Between September and January, the activity (nmol l(-1) h(-1)) of this size class was mostly supported by the low affinity component. The specific activity (nmol l(-1) h(-1) microg(-1)) of the high affinity component was highest in June and August. No significant correlation was found between phosphatase activities and chlorophyll a or total cell abundance. In return temporary relationships with specific taxa exist in particular with Ceratium spp., Gymnodinium spp. and Protoperidinium spp. The contribution of the 0.25-5 microm size class exceeded rarely 20% of the total particulate activity. Between June and August, high specific activities (nmol l(-1) h(-1) microg(-1)) were observed for its high affinity component. In autumn, strong rainfall increased the phosphate and nitrate concentrations and led to a drop in salinity, which probably explains the low phosphatase activities (nmol l(-1) h(-1)) and cell densities observed during this period.     

Development of hepatic CYP1A and blood vitellogenin in eel (Anguilla anguilla) for use as biomarkers in the Thames Estuary, UK.

The potential of eel (Anguilla anguilla) as a monitoring species for the Thames Estuary, UK, was examined. Hepatic cytochrome P4501A [7-ethoxyresorufin O-deethylase (EROD) activity] and blood vitellogenin (Western analysis) were investigated as biomarkers of exposure to, respectively, organic contaminants and to contaminants showing estrogenic activity. Hepatic microsomal EROD activities in A. anguilla from seven sites in the Thames Estuary in May 1998 varied three-fold (111 +/- 24 to 355 +/- 42 pmol min-1 mg protein-1) (mean +/- S.E.M.) and showed correlation with salinity; however, the latter relationship was not maintained at other times of the year. The range of EROD activities was two- to eight-fold higher than the 37 +/- 8 pmol min-1 mg-1 for A. anguilla from the relatively clean Tamar Estuary. beta-Naphthoflavone treatment (5 mg kg-1 wet wt.; 2 days) of Thames A. anguilla produced a two-fold increase in hepatic microsomal EROD activity. Comparing the Thames EROD data with those for A. anguilla from well-characterised contaminated sites in the Netherlands (Van der Oost, R., Goks?yr, A., Celander, M., Heida, H., & Vermeulen, N. P. E. 1996. Aquatic Toxicology, 36, 189-222), the Thames is suggested to be moderately impacted by polycyclic aromatic hydrocarbons and related contaminants. 17-beta-Estradiol treatment produced the appearance of a plasma protein of 211 Kd app. mol. wt. (recognised by antibodies to vitellogenin of Morone saxatilis), but putative vitellogenin could not be detected in A. anguilla from selected sites in the Thames Estuary.     

Correlative changes in metabolism and DNA damage in turbot (Scophthalmus maximus) exposed to benzo[a]pyrene

Juvenile turbot (Scophthalmus maximus) were injected intraperitoneally with either corn oil or 5 mg/kg benzo[a]pyrene (BaP) dissolved in corn oil and sampled I and 3 days after injection. After 1 day, no elevation of 7-ethoxyresorufin O-deethylase (EROD) activity was observed, however bile metabolites (BaP-7,8 dihydrodiol representing 70% of the total metabolites) and a single hepatic DNA adduct spot (0.47 adducts/10(8) nucleotides) identified by 32P-postlabelling were formed. No BaP metabolites or DNA adducts were observed in either control or carrier control fish. Fish sampled after 3 days reported 5-fold higher (P < 0.05) levels of EROD activity, a shift in the bile metabolite profile towards BaP phenol formation (1OH and 30H BaP comprising up to 60% of total metabolites detected) and the formation of two adduct spots (0.86 and 0.71 adducts/10(8) nucleotides). These results show that BaP can be metabolised and form hydrophobic DNA adducts in turbot without EROD elevation. Following EROD elevation, a shift in the profile of both BaP metabolites and BaP metabolite-DNA interactions occurs indicative of other oxidative processes.     

A piscine glutathione S-transferase which efficiently conjugates the end-products of lipid peroxidation

A cDNA clone for glutathione S-transferaseA (GSTA) from plaice (Pleuronectes platessa) was expressed in Eschericia coli (E. coli) and purified to homogeneity by S-hexylglutathione affinity chromatography. When compared to literature values for a variety of purified mammalian GSTs, the heterologously expressed purified plaice enzyme had moderate activity towards the model substrate 1,2-chloro-2,4-dinitrobenzene (CDNB) and exhibited a Km of 2.5 ± 2 mM and Vmax of 30.9 ± 2.3 μmol min⁻¹ mg⁻¹. It had little or no activity towards several other model GST substrates including 1,2-dinitrochloro-4-benzene (DCNB), ethacrynic acid (EA), and p-nitrobenzylchloride (NBC). However plaice GSTA was a relatively efficient catalyst for the conjugation of a series of alk-2-enals and alk-2,4-dienals and also 4-hydroxynonenal. The highest activity observed with this series of substrates was with trans-non-2-enal with a Km of 17.9 ± 2.2 μM and a Vmax of 3.01 ± 0.57 μmol min⁻¹ mg⁻¹. These unsaturated alkenals have been identified in cells and cell extracts as highly toxic products arising from peroxidation of unsaturated fatty acids particularly during periods of oxidative stress. Fish are relatively rich in polyunsaturated fatty acids and thus GSTA mediated conjugation may be an important mechanism for detoxifying peroxidised lipid breakdown products.     

In vitro mechanistic differences in benzo[a]pyrene-DNA adduct formation using fish liver and mussel digestive gland microsomal activating systems

Turbot (Scophthalmus maximus) and mussel (Mytilus edulis) microsomes were incubated with DNA to examine if microsomal in vitro metabolism of BaP could result in DNA adducts detected by 32P-postlabelling. Turbot DNA was incubated with benzo[a]pyrene (BaP), NADPH and microsomal activating systems prepared from either livers of unexposed turbot, turbot exposed to BaP or beta-naphthoflavone (beta-NF) or digestive glands from mussels. The beta-NF activating system generated the highest levels of DNA adducts detected in this study (451.7 adducts per 10(8) nucleotides) and were distributed in three discrete adduct TLC spots, one of which (97% of the total adducts) co-migrated with the 32P-postlabelled BaP 7,8-diol, 9,10-epoxide-N2-guanine adduct. Fewer adducts (P < 0.05) were generated by BaP-induced microsomes (9.4-30.6 adducts per 108 nucleotides) but levels were higher (P <0.05) than those generated from untreated fish (3.5 adducts per 10(8) nucleotides). Co-incubation with 500 microM alpha-naphthoflavone (alpha-NF) resulted in 97-99% inhibition in adduct formation implicating cytochrome P450-dependent (CYP) bioactivation however there was some evidence for carry over of BaP in the liver microsomal preparations from BaP injected fish. In contrast to the fish activating systems, no DNA adducts were observed when mussel microsomes were incubated with BaP, DNA and NADPH.     

Temporal variations in dimethylsulphoniopropionate and dimethyl sulphide in the Zuari estuary,Goa (India)

Despite tropical estuarine systems representing important sites for active biogeochemical processes, studies on dimethyl sulphide (DMS) in these systems are sparse. Here we report on DMS and dimethylsulphoniopropionate (DMSP) variability in relation to physicochemical and biological parameters for a period of 14 months in a tropical estuarine environment. DMS and DMSP showed high temporal variations with maximal concentrations during the southwest monsoon coinciding with a dinoflagellate bloom. Dinoflagellates appear to be the major contributors to the DMSP pool. Average DMS and DMSP concentrations (surface and bottom) suggested that much of the DMSP produced is converted to forms other than DMS. Surface DMS varied between 0.3 and 15.4 nmol dm(-3) while DMSP ranged from 0.8 to 419.5 nmol dm(-3). The DMS flux was 0.03-1.9 microM m(-2) d(-1) (average=0.6 microM m(-2) d(-1)) during the study period, that concurs well with the values reported for temperate estuaries.     

Metabolism of a model environmental carcinogen by bivalve molluscs

Bivalve molluscs have been used as monitors of marine pollution because they concentrate xenobiotics of many kinds in their tissues. Marine pollutants often produce stress responses such as reduced scope for growth, lysosomal destabilization, altered immune responsiveness and other sequelae. The ability of bivalves to metabolize organic compounds via mixed-function oxygenases (MFO) was demonstrated by the epoxidation of aldrin¹ and benzo[a]pyrene (BP);² this work has been corroborated and extended in many subsequent studies. Biotransformation of BP and other environmental procarcinogens can generate either activated or detoxified metabolites. In this paper the BP metabolites produced by digestive gland homogenates of the clam (Mercenaria mercenaria) and the oyster (Crassostrea virginica) are described. Control bivalves produced the proximate carcinogen (BP 7,8-dihydrodiol), as well as various minimally reactive BP diols and monohydroxylated metabolites. Seven days after injection of the potent inducer of mammalian MFO, Aroclor 1254, BP 7,8-diol production was augmented in Mercenaria, and atypical quinones and phenols were seen in both species. However, in the Ames bacterial mutagenicity assay, homogenates from Aroclor-treated clams failed to activate benzo[a]pyrene. The biological significance of BP metabolism in bivalves is unknown; however, it may prove to be a useful index of pollution.     

Origin and characteristics of the zooplankton phosphatase activity in a coastal ecosystem of the Mediterranean sea (Toulon Bay)

In Toulon Bay (France), very high phosphatase activities have been found in the zooplankton fraction>90 microm. This work was intended to specify their origin. For that purpose, larvae, juvenile and adult Crustacea (Copepods: Calanoids, Cyclopoids, Branchiopods: Cladocera, and Cirripeds) were isolated. Their activities were measured using paranitrophenyl phosphate dissolved in sea water in order to calculate Km (the enzyme half saturation concentration) and Vmax (the reaction rate when the enzyme is saturated with substrate). Vmax were referred to protein contents of the isolated organisms to calculate specific activities. For all zooplankton groups high and low affinity phosphatase activities were found. The low affinity enzyme was responsible for at least 70% of the total phosphatase activity. Its specific activity was higher for larvae than for copepodites and adults. In Cirriped nauplii this activity was particularly high with values which were several hundred times higher than that in other Crustacea. These enzymes had optimum pH close to 8.4, magnesium requirement and were competitively inhibited by orthophosphate. Experiments with intact and lysed Cirriped nauplii confirmed that living organisms had only a weak external activity and showed that most of the activity of these larvae was primarily intracellular.     

菲律宾蛤仔碱性磷酸酶的分离纯化及部分性质研究

经Tris-HCl缓冲液浸提、硫酸铵分级沉淀盐析、DEAE-Sepharose Fast Flow离子交换层析和Sephadex G-75凝胶过滤层析等步骤,从菲律宾蛤仔内脏团中分离得到碱性磷酸酶,SDS-PAGE显示为单一条带,提纯倍数为14.42,比活力达到131.08U/mg。酶学性质研究表明:该酶亚基分子量约44kD,最适反应温度为45℃,最适pH值为9.0,米氏常数Km为0.098mmol/L,最大反应速度Vmax为1.01μmol/(mL·min)。Ca2+、Mg2+对酶活力表现出显著的激活作用。     

节旋藻和螺旋藻对7种抗生素敏感性的比较研究

对两种丝状蓝藻(钝顶节旋藻和盐泽螺旋藻)在基因工程中常用作选择试剂的7种抗生素——氯霉素、氨苄青霉素、红霉素、链霉素、卡那霉素、庆大霉素和新霉素的敏感性作比较实验.结果表明,两种蓝藻对抗生素的敏感性既有共同的特点,也有明显的差异.它们对红霉素、氯霉素和链霉素最敏感,致死浓度分别为0.1,0.5和5μg/cm3.两种蓝藻对氨苄青霉素比较敏感,1μg/cm3的氨苄青霉素即可抑制Arthrospira.341和Spirulina.351的生长,但6d后生长恢复.Arthrospira.341和Spirulina.351对卡那霉素、庆大霉素和新霉素均有抗性,而且存在很大差异:300μg/cm3的卡那霉素对Arthrospira.341的生长仍然没有影响,但对于Spirulina.351,50μg/cm3的卡那霉素即对其生长有明显抑制作用;200μg/cm3的卡那霉素即可将其全部致死.200μg/cm3的庆大霉素和300μg/cm3的新霉素不能抑制Arthrospira.341和Spirulina.351的生长,但在这两种抗生素环境中两种藻的生长状态有很大差异.并验证了氯霉素、红霉素和链霉素是节旋藻和螺旋藻基因转化过程中的有效的抗性选择剂,也从对抗生素敏感性方面表明节旋藻和螺旋藻两个属的遗传差异.     

Effects of waterborne nitrite onphase I–II biotransformation in channel catfish(Ictalurus punctatus)

The effects of waterborne nitrite (3 mg/l NO₂) on channel catfish were studied to evaluate changes in hematological parameters and phase I–II biotransformation in liver slices. Nitrite-exposed fish had significantly higher methemoglobin, blood and liver nitrite, and significantly lower pO₂ than control fish. Total phase I-mediated metabolism of 7-ethoxycoumarin (EC) was not altered in nitrite-exposed fish compared with control fish (291±43 and 312±20 pmol/mg/h, respectively). However, phase II glucuronosyltransferase-mediated metabolism of 7-hydroxycoumarin (HC), both as a phase I metabolite of EC and as a parent substrate, was elevated in nitrite-exposed fish (204±17 and 1007±103 pmol/mg/h, respectively) as compared to control fish (149±14 and 735±87 pmol/mg/h) (P<0.05). Sulfotransferase-mediated metabolism of HC (as a metabolite of EC and as a parent substrate) was not notably altered in nitrite-exposed fish (95±16 and 617±33 pmol/mg protein/h, respectively) as compared with control fish (118±24 and 575±55 pmol/mg/h, respectively). These studies indicate that in vivo nitrite exposure and associated changes in hematological parameters do not appear to affect hepatic phase I EC biotransformation in channel catfish. However, subtle but significant changes in phase II glucuronidation, but not sulfation activity, were observed. The mechanism of these alterations is unclear. However, the data suggest that environmentally realistic concentrations of nitrite may affect the dynamics of conjugative metabolism in exposed fish.     

Altered glutathione S-transferase catalytic activities in female brown bullheads from a contaminated central Florida lake.

Brown bullheads (Ameriurus nebulosus) are a demersal freshwater species that can be found in a number of polluted ecosystems. The purpose of the present study was to determine the overall capacity for in vitro glutathione S-transferase (GST) detoxification by brown bullheads, and to see if bullhead GST catalysis was altered in bullheads from a polluted site. Brown bullhead liver cytosolic GSTs catalyzed the conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) over a large range of substrate concentrations, with apparent Km and Vmax for CDNB at fixed nucleophile (glutathione, GSH) concentrations of 1.8-1.9 mM and 12.1-14.6 mumol CDNB conjugated/min/mg, respectively. Bullhead GSTs were also highly active toward other substrates such as ethacrynic acid (ECA), delta 5-androstene-3,17-dione (ADI), and nitrobutyl chloride (NBC). Initial rate GST catalytic activities toward CDNB, NBC, ECA, and ADI were significantly lower in female bullheads from a contaminated lake (Lake Apopka Marsh) as compared to female bullheads inhabiting a nearby control site (Lake Woodruff). No site differences were observed with respect to male bullhead GST activities. These studies suggest that brown bullheads efficiently carry out GST conjugation of diverse electrophilic substrates. However, bullhead GST catalysis may be compromised in bullheads inhabiting polluted ecosystems.     

Effects of waterborne nitrite on phase I-II biotransformation in channel catfish (Ictalurus punctatus).

The effects of waterborne nitrite (3 mg/l NO2) on channel catfish were studied to evaluate changes in hematological parameters and phase I-II biotransformation in liver slices. Nitrite-exposed fish had significantly higher methemoglobin, blood and liver nitrite, and significantly lower pO2 than control fish. Total phase I-mediated metabolism of 7-ethoxycoumarin (EC) was not altered in nitrite-exposed fish compared with control fish (291 +/- 43 and 312 +/- 20 pmol/mg/h, respectively). However, phase II glucuronosyltransferase-mediated metabolism of 7-hydroxycoumarin (HC), both as a phase I metabolite of EC and as a parent substrate, was elevated in nitrite-exposed fish (204 +/- 17 and 1007 +/- 103 pmol/mg/h, respectively) as compared to control fish (149 +/- 14 and 735 +/- 87 pmol/mg/h) (P < 0.05). Sulfotransferase-mediated metabolism of HC (as a metabolite of EC and as a parent substrate) was not notably altered in nitrite-exposed fish (95 +/- 16 and 617 +/- 33 pmol/mg protein/h, respectively) as compared with control fish (118 +/- 24 and 575 +/- 55 pmol/mg/h, respectively). These studies indicate that in vivo nitrite exposure and associated changes in hematological parameters do not appear to affect hepatic phase I EC biotransformation in channel catfish. However, subtle but significant changes in phase II glucuronidation, but not sulfation activity, were observed. The mechanism of these alterations is unclear. However, the data suggest that environmentally realistic concentrations of nitrite may affect the dynamics of conjugative metabolism in exposed fish.