Pentoxyresorufin O-deethylase Research Articles

Biotransformation and oxidative stress responses in relation to tissue contaminant burden in Clarias gariepinus exposed to simulated leachate from a solid waste dumpsite in Calabar, Nigeria

In this study, we have investigated biotransformation and oxidative stress responses in relation to tissue contaminant burden in the African sharptooth catfish (Clarias gariepinus) exposed to simulated leachate from a solid waste dumpsite in Calabar, Nigeria. Fish were exposed to simulated leachate, diluted to 0:0 (negative control), 1:10, 1:50, 1:100 and phenanthrene (a PAH: 50 μg/L used as a positive control) for 3, 7 and 14 days. Hepatic transcripts for cat, sod1, gpx1, gr, gst, cyp1a, cyp2d3, and cyp27 were analyzed by real-time PCR, while enzymatic assays for ethoxyresorufin O-deethylase (EROD), buthoxyresorufin O-deethylase (BROD), methoxyresorufin O-deethylase (MROD), pentoxyresorufin O-deethylase (PROD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione S-transferase (GST), uridine diphospho-glucuronosyltransferase (UDPGT) and lipid peroxidase (LPO) were measured using standard methods. In addition, protein expression for CYP1A, CYP3A and metallotheionin (MT) were measured by immunoblotting. Fish muscle samples were analyzed for selected group of contaminants after 14 days exposure showing significantly high uptake of heavy metals (Cd, Hg and Pb), polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), chlorophenols, organochlorine (OC) and organophosphate pesticides in exposed fish. We observed significant concentration- and time-specific increases in biotransformation and oxidative stress responses at transcript and functional (enzyme and protein) levels, that paralleled tissue contaminants bioaccumulation patterns, after exposure to the simulated leachates. Our results highlighted the potential environmental, wildlife and public health consequences from improper solid waste disposal. In addition, it also provides a scientific basis for local sensitization and inform legislative decisions and policy formulation towards sustainable environmental management of solid wastes in Nigeria and other developing countries.

Effects of atrazine and chlorpyrifos on cytochrome P450 in common carp liver

Atrazine (ATR) and chlorpyrifos (CPF), widely used in agriculture, have resulted in a series of toxicological and environmental problems. We investigated the activities of the biotransformation enzymes ethoxyresorufin-O-deethylase (EROD) and pentoxyresorufin-O-deethylase (PROD), total cytochrome P450 (CYP), CYP1A mRNA level and level of tissue ATR, CPF, and their metabolites in the liver of common carp (Cyprinus carpio L.) after a 40-d exposure to CPF and ATR, alone or in combination, and a 20-d recovery. In the present study, juvenile common carp was exposed to ATR (at concentrations of 4.28, 42.8 and 428μgL−1), CPF (1.16, 11.6 and 116μgL−1), and ATR/CPF mixture (at concentrations of 1.13, 11.3 and 113μgL−1). A general increasing trend for the activity of the biotransformation enzymes (EROD and PROD), CYP and CYP1A mRNA level was observed in the liver of common carp exposed to ATR, CPF and the ATR/CPF mixture. In addition, ATR, CPF, and their metabolites demonstrated a high accumulation in the liver. These results demonstrated that the CYP system in fish could be used as a biomarkers in evaluating the impact of ATR and CPF exposure on the common carp.

Are deep-sea organisms dwelling within a submarine canyon more at risk from anthropogenic contamination than those from the adjacent open slope? A case study of Blanes canyon (NW Mediterranean)

Due to their geomorphological structure and proximity to the coastline, submarine canyons may act as natural conduit routes for anthropogenic contaminants that are transported from surface waters to the deep-sea. Organisms dwelling in these canyon environments might thus be at risk of experiencing adverse health effects due to higher pollution exposure. To address this question, chemical and biochemical analyses were conducted on two of the most abundant deep-sea fish species in the study area, namely Alepocephalus rostratus and Lepidion lepidion, and the most abundant deep-sea commercial decapod crustacean Aristeus antennatus sampled inside Blanes canyon (BC) and on the adjacent open slope (OS). Persistent organic pollutants (POPs) levels, including polychlorinated biphenyl (PCB), dichlorodiphenyltrichloroethane (DDT) and derivatives, hexachlorocyclohexanes (HCHs) and hexachlorobenzene (HCB) were determined in muscle tissue of selected samples from 900m and 1500m depth. Potential effects resulting from contaminant exposure were determined using hepatic biomarkers such as ethoxyresorufin-O-deethylase (EROD), pentoxyresorufin-O-deethylase (PROD), catalase (CAT), carboxylesterase (CbE), glutathione-S-transferase (GST), total glutathione peroxidase (GPX), glutathione reductase (GR) and superoxide-dismutase (SOD) enzyme activities and lipid peroxidation levels (LP). L. lepidion and A. antennatus tissues exhibited higher POP levels inside BC compared to the OS at 900m depth. These findings were consistent with biomarker data (i.e. enzymatic response to presence of contaminant agents). Elevated xenobiotic-metabolizing (EROD and PROD) and antioxidant enzymes (CAT and GPX) indicated higher contaminant exposure in both species caught within BC. No difference in POP accumulation between sites was observed in L. lepidion at 1500m depth, nor in biomarker data, suggesting that the pollution gradient was less pronounced at greater depths. This trend was further corroborated by the results obtained for A. rostratus at 1500m depth. Hence, the present findings suggest the, at least temporary, existence of a pollution gradient between Blanes canyon and the open slope at shallower depths and this resulted in alterations of the physiology of deep-sea organisms dwelling within this area.

Cytochrome P450 system as potential biomarkers of certain toxicants: comparison between plant and animal models

In present study, we measured the activities of some selected cytochrome P450 isozymes like ethoxyresorufin-o-deethylase (EROD), pentoxyresorufin-o-deethylase (PROD), and N-nitrosodimethylamine demethylase (NDMA-d) treating experimental rats with different environmental toxicants, namely trichloroethylene (TCE), heavy metal mixture, and ethyl alcohol individually or in combination and by exposing the Allium cepa bulbs to increasing concentration of TCE for 48 h. In animal system, NDMA-d activity displayed a remarkable amplification by 4.2-fold in the liver of alcohol ingested rats compared with control animals. TCE intake also resulted in a marked increase of around 3.8-fold and 1.2-fold in rats' NDMA-d activity in the liver and kidney, respectively. In onion bulbs, an amazing rise in the activity of test enzymes was recorded in a dose-dependent manner. Among the said enzymes, PROD showed maximum increase up to the extent of 22-fold in comparison with control at 20 ppm of TCE exposure, whereas NDMA-d and EROD showed 11- and 9-fold increase in enzymatic activity, respectively, compared with the control. Based on this study, we conclude and advocate that all the selected isozymes of CYP450 system, viz. PROD, NDMA-d, and EROD can act as potent biomarkers in plant system for assessing the TCE pollution.

Natural variability of hepatic biomarkers in Mediterranean deep-sea organisms

Biomarker assays are widely used as proxies for contaminant-induced effects in aquatic organisms. However, in many cases, their intrinsic natural variability due to exogenous and endogenous factors makes the interpretation of biomarker data difficult. In the present study, we investigated the natural fluctuations of six hepatic biomarkers, namely ethoxyresorufin-O-deethylase (EROD) in fish and pentoxyresorufin-O-deethylase (PROD) in crustacea, catalase (CAT), carboxylesterase (CbE), glutathione-S-transferase (GST), total glutathione peroxidase (GPX) and glutathione reductase (GR) in two deep-sea fish species, namely Alepocephalus rostratus and Lepidion lepidion and the decapod crustacean Aristeus antennatus. The NW Mediterranean deep-sea environment is characterized by very stable temperature and salinity conditions, allowing the exclusion of these two factors as potential sources of interference with biomarker activities. Biomarker results exhibited a clear influence of reproductive processes on enzyme activities, in particular in A. rostratus, which presented a pronounced seasonal pattern linked to variations in the gonadosomatic index (GSI). In addition, other factors such as food availability may also have influenced the observed variability, in particular in specimens of L. lepidion, which did not exhibit variations in reproductive activity throughout the sampling period. Depth-related variability did not exhibit a clear trend and fluctuations across sampling depths were not attributable to any specific factor. Body size had also a significant influence on some biomarkers, although allometric scaling of certain enzyme activities appears to be species-specific. The present work has thus shown that despite the lack of fluctuations of abiotic parameters such as temperature and salinity, biomarker activities in deep-sea organisms still exhibit significant variability, mainly as a result of reproductive processes and food availability.

Tissue specificity of aryl hydrocarbon receptor (AhR) mediated responses and relative sensitivity of white sturgeon (Acipenser transmontanus) to an AhR agonist

Sturgeons are endangered in some parts of the world. Due to their benthic nature and longevity sturgeon are at greater risk of exposure to bioaccumulative contaminants such as dioxin-like compounds that are associated with sediments. Despite their endangered status, little research has been conducted to characterize the relative responsiveness of sturgeon to dioxin-like compounds. In an attempt to study the biological effects and possible associated risks of exposure to dioxin-like compounds in sturgeon, the molecular and biochemical responses of white sturgeon (Acipenser transmontanus) to a model aryl hydrocarbon receptor (AhR) agonist, β-naphthoflavone (βNF) were investigated. White sturgeon were injected intraperitoneally with one of three doses of βNF (0, 50, or 500mg/kg, bw). Rainbow trout (Oncorhynchus mykiss) were used as a reference species since their responses have been well characterized in the past. Three days following injection with βNF, fish were euthanized and livers, gills, and intestines collected for biochemical and molecular analyses. White sturgeon exposed to βNF had significantly greater ethoxyresorufin O-deethylase (EROD) activity in liver (up to 37-fold), gill (up to 41-fold), and intestine (up to 36-fold) than did unexposed controls. Rainbow trout injected with βNF exhibited EROD activity that was significantly greater in liver (88-fold), than that of controls, but was undetectable in gills or intestine. Abundance of CYP1A transcript displayed a comparable pattern of tissue-specific induction with intestine (up to 189-fold), gills (up to 53-fold), and liver (up to 21-fold). Methoxyresorufin O-deethylase (MROD) and pentoxyresorufin O-deethylase (PROD) activities were undetectable in unexposed white sturgeon tissues while exposed tissues displayed MROD activity that was only moderately greater than the activity that could be detected. Differential inducibility among liver, gill, and intestine following exposure to an AhR agonist is likely associated with tissue-specific regulation of the AhR signalling pathway. Liver and gill of white sturgeon had significantly greater AhR transcript abundance than did the intestine, however following exposure to βNF, significantly greater induction in AhR transcript abundance was detected in intestine (up to 35-fold) compared to liver (up to 5-fold) or gills (up to 11-fold). It was shown that white sturgeon are responsive to AhR agonists in the liver, gill, and intestine and could be among the more sensitive fish species with regard to inducibility of CYP1A.

Alterations in immune function and CYP450 activity of adult male deer mice following exposure to either 7,12-dimethylbenz[a]anthracene or benzo[b]fluoranthene

Adult male deer mice were exposed every other day for a period of 11 days to either 7,12-dimethylbenzanthracene (DMBA; CAS# 57-97-6) or benzo[b]fluoranthene (BbF; CAS# 205-99-2) (0, 0.3, 1, 3, 10, or 30 mg kg−1). Immune endpoints assessed were lymphocyte proliferation, macrophage pinocytosis, and the antibody plaque-forming cell (PFC) response. Cytochrome P450 (CYP450) activity was assessed using ethoxyresorufin-O-deethylase (EROD) and pentoxyresorufin-O-deethylase (PROD). Macrophage pinocytosis was not altered by either compound. Both T- and B-cell proliferations were significantly increased by DMBA at 0.3 or 1 mg kg−1 and by BbF at 10 or 30 mg kg−1, but decreased by DMBA at 30 mg kg−1. Sheep red blood cell (SRBC)-specific-IgM production, as measured by the PFC response, was the most striking adverse immune effect observed and was significantly suppressed compared to control at all treatment concentrations for both compounds. EROD activity was markedly induced by DMBA at 30 mg kg−1, while BbF produced induction at 1, 10, or 30 mg kg−1. No marked effect on PROD activity was noted following DMBA treatment, but BbF-induced PROD activity at 1, 10, or 30 mg kg−1. Unexpectedly, four of six mice in the 30 mg DMBA kg−1 group did not survive to the end of the experiment, and one animal died in both the 3 and 10 mg kg−1 treatments. The calculated LD50 was 20.8 mg DMBA kg−1. The PFC response in deer mice was a more sensitive endpoint than CYP450 activity, suggesting that utilization of CYP450 endpoints in risk assessment without assessment of immune function, specifically antibody production, might possibly underestimate the risk to wild rodents environmentally exposed to polycyclic aromatic hydrocarbons.

Effects of Polycyclic Aromatic Hydrocarbons in Northern Bobwhite Quail (Colinus virginianus)

Polycyclic aromatic hydrocarbons (PAH) are ubiquitous contaminants of aquatic and terrestrial ecosystems, and are known to induce biochemical alterations in exposed organisms. Aside from a variety of adverse physiological effects associated with exposure to petroleum products, oils, and oil sludges, little is known about the effects of individual PAH on birds. Acute toxicity of naphthalene, pyrene, and benz[a]anthracene (BAA) was examined in adult northern bobwhite quail (Colinus virginianus). Additionally, subacute (8 d) and subchronic (60 d) studies were conducted to assess alterations in metabolic enzyme activity. Neither naphthalene, nor pyrene, nor BAA exposure via oral gavage produced acute toxicity up to the limit dose of 2 g/kg body weight. In the subacute study, quail provided feed containing the highest concentration of BAA for 5 d had significantly increased renal ethoxyresorufin O‐deeththylase (EROD) activity compared to controls. Following a 3-d recovery period, significant increases between 10 and 100 mg/kg of BAA in feed existed for both hepatic EROD and pentoxyresorufin O-deethylase (PROD) activity compared to controls. Subchronic exposure to BAA (ranging from 0.1 to 10 mg/kg) also resulted in a significant rise of EROD and PROD in both kidney and liver tissue compared to controls. Though the individual PAH used in this study were not acutely toxic, these results confirm that these individual PAH induce alterations in metabolic enzyme activity in northern bobwhite quail.

Cytochrome P450 system as a toxicity biomarker of industrial wastewater in rat tissues

In the present study the effect of laboratory exposure to wastewaters from Aligarh (AWW) and Saharanpur (SWW) on the activities of cytochrome P450 (CYP450) isozymes like ethoxyresorufin-o-deethylase (EROD), pentoxyresorufin-o-deethylase (PROD) and N-nitrosodimethylamine demethylase (NDMA-d) were investigated in the liver and kidney of rats. The industrial wastewater samples from Saharanpur city of northern India resulted 12, 3.5 and 1.5-fold rise in the EROD (CYP1A1), PROD (CYP2B1) and NDMA-d (CYP2E1) activity, respectively, in the liver of treated animals. Renal EROD and PROD activities were found to be enhanced by around 5 and 7-folds, respectively, as a result of SWW treatment. On the other hand, Aligarh samples showed significant inhibition in these test CYP450 enzymes both in hepatic as well as renal tissues. Strong induction of CYP1A1 (>10-fold) suggests that EROD can serve as a potent biomarker of SWW in the liver of treated animal. However, PROD and EROD can also act as fairly good biomarkers in case of renal tissue. Marked elevation of EROD activity in SWW treated animals strongly suggests the overwhelming levels of EROD inducers in the Saharanpur sample while a meagre amount of inducers accompanied with significant levels of inhibitors in the Aligarh sample.

Evaluating the impact of environmental pollution on fish in McMurdo Sound, Antarctica: A biomarker approach

To assess the ongoing effects of anthropogenic pollutants on the Winter Quarters Bay (WQB) fauna, resident Trematomus hansoni and T. hansoni from a control site, Backdoor Bay (BDB) were caught. A suite of biomarkers, namely ethoxyresorufin- o-deethylase (EROD), pentoxyresorufin- o-deethylase (PROD), as well as biliary naphthalene and phenanthrene were measured. EROD activity was significantly higher in the WQB resident T. hansoni, while the remaining parameters were unaffected, and no differences in the hepatosomatic index or condition factor were evident. Additionally, Trematomus bernacchii were collected from BDB and placed in cages within WQB and at Cape Armitage (another control site near McMurdo Station) for 2 and 4 weeks. No differences in EROD and PROD activities were found between any of the caged T. bernacchii. Naphthalene was significantly elevated in WQB caged T. bernacchii after both 2 and 4 weeks even though phenanthrene was elevated in WQB caged fish only after 4 weeks. Again, there were no significant morphological differences between groups. It is evident from both the resident and caged fish that the legacy left by the pollution in WQB is still affecting the local marine fauna.

Short-term Exposure to Triclosan Decreases Thyroxine In Vivo via Upregulation of Hepatic Catabolism in Young Long-Evans Rats

Triclosan (5-chloro-2-(2,4-dichlorophenoxy)-phenol) is a chlorinated phenolic antibacterial compound found in consumer products. In vitro human pregnane X receptor activation, hepatic phase I enzyme induction, and decreased in vivo total thyroxine (T4) suggest adverse effects on thyroid hormone homeostasis. Current research tested the hypothesis that triclosan decreases circulating T4 via upregulation of hepatic catabolism and transport. Weanling female Long-Evans rats received triclosan (0-1000 mg/kg/day) by gavage for 4 days. Whole blood and liver were collected 24 h later. Total serum T4, triiodothyronine (T3), and thyroid-stimulating hormone (TSH) were measured by radioimmunoassay. Hepatic microsomal assays measured ethoxyresorufin-O-deethylase, pentoxyresorufin-O-deethylase (PROD), and uridine diphosphate glucuronyltransferase enzyme activities. The messenger RNA (mRNA) expression of cytochrome P450s 1a1, 2b1/2, and 3a1/23; UGTs 1a1, 1a6, and 2b5; sulfotransferases 1c1 and 1b1; and hepatic transporters Oatp1a1, Oatp1a4, Mrp2, and Mdr1b was measured by quantitative reverse transcriptase PCR. Total T4 decreased dose responsively, down to 43% of control at 1000 mg/kg/day. Total T3 was decreased to 89 and 75% of control at 300 and 1000 mg/kg/day. TSH did not change. Triclosan dose dependently increased PROD activity up to 900% of control at 1000 mg/kg/day. T4 glucuronidation increased nearly twofold at 1000 mg/kg/day. Cyp2b1/2 and Cyp3a1/23 mRNA expression levels were induced twofold and fourfold at 300 mg/kg/day. Ugt1a1 and Sult1c1 mRNA expression levels increased 2.2-fold and 2.6-fold at 300 mg/kg/day. Transporter mRNA expression levels were unchanged. These data denote important key events in the mode of action for triclosan-induced hypothyroxinemia in rats and suggest that this effect may be partially due to upregulation of hepatic catabolism but not due to mRNA expression changes in the tested hepatic transporters.

Aroclor 1254 induced cytotoxicity and mitochondrial dysfunction in isolated rat hepatocytes

Polychlorinated biphenyls (PCBs) are widespread persistent environmental contaminants that display a complex spectrum of toxicological properties, including hepatotoxicity. Although Aroclor 1254 is ubiquitous in the environment, its potential cytotoxic effect on rat hepatocytes and the mechanism underlines its cytotoxicity are not fully investigated. Therefore, the present study was conducted to investigate: (1) the potential cytotoxicity of Aroclor 1254 in rat hepatocytes, and (2) characterization of the molecular mechanisms involved in the Aroclor 1254-induced hepatotoxicity, particularly the role of mitochondria, possibly a primary target in such event, could greatly explain the cytotoxic effect of Aroclor 1254 in rat hepatocytes. Hepatocytes were isolated from adult male albino rats and incubated for 24 h in a fresh media containing 0, 20, 30, 40, 50 or 60 μM of Aroclor 1254. At the end of incubation, hepatocytes and hepatocyte mitochondria were used for the assay. Our results showed cytotoxicity of Aroclor 1254 in rat hepatocytes starting at a concentration of 30 μM as manifested by increased lactate dehydrogenase (LDH) leakage, decreased cell viability (MTT assay) and increased lipid peroxidation. As mitochondria are known to be one possible site of the cell damage, the effects of Aroclor 1254 on hepatocyte mitochondria was investigated. Aroclor 1254 induced reactive oxygen species (ROS) generation in hepatocyte mitochondria, inhibited mitochondrial respiratory chain complexes I and III and β-oxidation of free fatty acids, depletion of mitochondrial antioxidant enzymes GPx and GR and the non-enzymatic antioxidant reduced glutathione, inhibited mitochondrial membrane potential (Δψ m), decreased mitochondrial aconitase and cardiolipin content, and elevated levels of cytochrome P450 subfamily, CYP1A and CYP2B activities as indicated by ethoxyresorufin O-deethylase (EROD) and pentoxyresorufin O-deethylase (PROD). Therefore, we can conclude that Aroclor 1254 induced rat hepatocyte toxicity and our findings provide evidence to propose that mitochondria are one of the most important and earliest cell targets in Aroclor 1254-mediated toxicity and delineate several mitochondrial processes at least, in part, by induction of oxidative stress. These findings can be useful in future cytoprotective therapy approaches. Since mitochondrial events appear to be targeted in hepatocellular damage induced by Aroclor 1254, an antioxidant therapy targeted to mitochondria may constitute an interesting strategy to ameliorate its toxicity.

Effects of Tributyltin (TBT) on In Vitro Hormonal and Biotransformation Responses in Atlantic Salmon (Salmo salar)

The mechanisms by which the biocide tributyltin (TBT) and its metabolites affect the hormonal and xenobiotic biotransformation pathways in aquatic species are not well understood. In this study hepatocytes isolated from salmon were used to evaluate the mechanistical effects of TBT on fish hormonal and xenobiotic biotransformation pathways. Cells were exposed to 0.01, 0.1, 1, or 5 μM TBT and samples were collected at 0, 12, 24, or 48 h following exposure. Gene expression patterns were evaluated using quantitative polymerase chain reaction (PCR), and cytochrome P-450 (CYP)-mediated enzyme activities were evaluated by ethoxyresorufin, benzyloxyresorufin, and pentoxyresorufin O-deethylase (EROD, BROD, and PROD, respectively) activity assays. Generally, exposure of hepatocytes to 1 μM (at 48 h) and 5 μM TBT (at 12, 24, and 48 h) consistently produced reductions in all mRNA species investigated. TBT produced significant decreases of vitellogen (Vtg) expression at 48 h and modified the expression patterns of estrogen receptors (ERα and ERβ) and androgen receptor-β (ARβ) that were dependent on time and TBT concentration. In the xenobiotic biotransformation pathway, TBT produced differential expression patterns that were dependent on exposure time and concentration for all salmonid AhR2 isoforms (AhR2α, AhR2β, AhR2δ, and AhR2γ). For CYP1A1, CYP3A, AhRR, and Arnt mRNA, TBT produced exposure- and time-specific modulations. Catalytic CYP activities showed that BROD activity increased in an apparent concentration-specific manner in cells exposed to TBT for 12 h. Interestingly, EROD activity showed a TBT concentration-dependent increase at 24 h and PROD at 12 and 48 h of exposure. In general our data show that TBT differentially modulated hormonal and biotransformation responses in the salmon in vitro system. The apparent and consistent decrease of the studied responses with time in 1 and 5 μM exposed hepatocytes suggest a possible transcription inhibitory effect of TBT.

Interaction of Sanguinarine Alkaloid, Isolated From Argemone Oil, With Hepatic Cytochrome P450 in Rats

ABSTRACTPrior studies have shown that argemone oil (AO), responsible for ‘Epidemic dropsy’, causes inhibition of catalytic activities of Cytochrome P450 (P450). In this study interaction of sanguinarine (SAN) alkaloid, isolated from AO, with rat hepatic P450 was investigated. Hepatic microsomes prepared from 3-methylcholantherene (3MC) treated rats when incubated with SAN (1–3 μM) resulted in a spectral peak at 385 nm and a trough at 415 nm, indicative of Type I binding. Incubation of SAN (50–200 μM) with hepatic microsomes prepared from phenobarbitone (PB) treated rats also showed a Type I spectra with a peak at 395 nm and a trough at 420 nm. Relative binding efficiency (ΔAmax/Ksapp factor) of SAN with P450 was found to be 1540 and 1030 absorbance units/nmol CYP/M for 3MC and PB induced microsomes, respectively. In a P450 spectral inhibition study SAN showed higher affinity towards 3MC eliciting inhibition at much lesser concentrations (0.25–5 μM) as compared to PB (100–300 μM). The IC50s of SAN with different catalytic markers of P450 isoforms, i.e. ethoxyresorufin-O-deethylase (EROD) for CYP1A1, was 2.8 μM and for methoxyresorufin-O-deethylase (MROD) for CYP1A2 was 2.2 μM in 3MC induced microsomes, while benzoyloxyresorufin-O-deethylase (BROD) for CYP 2B1/1A1 showed an IC50 of 50 μM but pentoxyresorufin-O-deethylase (PROD) for CYP2B1 showed no inhibition even at higher concentrations of SAN (> 60 μM) in PB-induced microsomes. These results indicate that higher affinity of SAN binding towards the CYP1A family may have a role in SAN toxicity.

Effect of a PCB-based transformer oil on testicular steroidogenesis and xenobiotic-metabolizing enzymes

Pyralene is a PCB-based transformer oil with a unique PCB congener profile when compared to other mixtures. We studied the influence of Pyralene on testicular steroidogenesis and the status of xenobiotic-metabolizing enzymes in the testis and liver of rats during oral exposure (10 and 50 mg/kg body weight, p.o. daily for 1 week) and a 3-week post-treatment recovery period. As expected, Pyralene induced a rapid and sustained increase in mRNA transcripts for CYP1A1 and CYP2B1 in hepatocytes that was associated with a dramatic increase in ethoxyresorufin- O-deethylase (EROD) and pentoxyresorufin- O-deethylase (PROD) activities. Testicular androgenesis and the conversion of progesterone to testosterone in testicular microsomes were bidirectionally affected. An increase in these parameters was observed 24 h after the initial administration of Pyralene, followed by inhibition that lasted until the fourth post-treatment day. Expression PCR analysis revealed a significant decrease in 17β-hydroxysteroid dehydrogenase (17βHSD) transcript abundance at 48 h after Pyralene administration. In contrast, transcripts for several other steroidogenic enzymes and for testicular CYP1A1, CYP1B1, and CYP2B1 were unaffected under the same conditions. These results in the rat indicate that a sub-chronic exposure to Pyralene disrupted testicular steroidogenesis and suggest the mechanism may involve direct action on the regulation of specific steroidogenic enzymes such as 17βHSD.

EFFECTS OF MOTORCYCLE EXHAUST INHALATION EXPOSURE ON CYTOCHROME P-450 2B1, ANTIOXIDANT ENZYMES, AND LIPID PEROXIDATION IN RAT LIVER AND LUNG

The effects of motorcycle exhaust (ME) on metabolic and antioxidant enzymes and lipid peroxidation were determined using male rats exposed to 1:10 diluted ME by inhalation 2h daily for 4wk. For microsomal cytochrome P-450 enzymes, ME resulted in threefold increases of 7ethoxyresorufin and pentoxyresorufin O-deethylase activities in liver and a sixfold increase of 7-ethoxyresorufin O-deethylase activity and an 80% decrease of pentoxyresorufin O-dealkylase activity in lung. The results of immunoblot analysis of microsomal proteins revealed that ME increased liver and lung cytochrome P-450 1A1 with minimal effects on cytochrome P-450 2E1. ME increased cytochrome P-450 2B1/2 proteins in liver but decreased cytochrome P-450 2B1 in lung. ME did not change microsomal cytochrome P-450 enzyme activity or protein level in kidney. For phase II enzymes, ME resulted in 53% and twofold increases of cytosolic NAD(P)H:quinone oxidoreductase activities in liver and lung, respectively, and no effect on microsomal UDP-glucuronosyltransferase activities. For antioxidant enzymes, ME produced 23% and 35% decreases of superoxide dismutase, 9% and 27% decreases of catalase, and no changes of glutathione peroxidase activities in liver and lung cytosols, respectively. For lipid peroxidation, the results of thiobarbituric acid assay showed that ME resulted in a twofold increase of formation of malondialdehyde by liver microsomes incubated with FeCl3-ADP. ME produced a threefold increase of malondialdehyde formation by lung microsomes. The present study demonstrates that ME inhalation exposure differentially modulates cytochrome P-450 2B1 and antioxidant enzymes and increases susceptibility to lipid peroxidation in rat liver and lung.

Dibromoacetic acid-induced elevations in circulating estradiol: effects in both cycling and ovariectomized/steroid-primed female rats

Oral exposures to high concentrations of the drinking water disinfection by-product dibromoacetic acid (DBA) over the course of 14 days have been found to disrupt estrous cyclicity in the female rat. In order to investigate possible alterations in the relevant hormonal regulatory mechanisms, female Sprague–Dawley rats were gavaged for 2 weeks with 270 mg/kg DBA, ovariectomized (OVX) and implanted with estradiol capsules. For these females, the induced luteinizing hormone (LH) surge in these animals showed a borderline suppression in peak LH concentrations that was accompanied by a marked increase in circulating estradiol. This elevation in estradiol was DBA dose-related and, for intact, normally cycling females receiving lower doses of DBA (60 and 120 mg/kg, 14 days), was present on the day of estrus, at a time when a dramatic fall from proestrous concentrations is normally evident. Evaluations of liver microsomal cytochrome P450 activity in OVX/estradiol-implanted rats showed a suppression in ethoxyresorufin- O-deethylase (EROD) and pentoxyresorufin- O-deethylase (PROD) activity (indications of the activity of CYP1A and 2B, respectively—two key enzymes in estradiol oxidative metabolism). Phenobarbital (PhB) exposure in these animals did show induction of this activity, but was unable to lower E2 concentrations. This suggests that a DBA-induced suppression in estradiol catabolism is present and may either involve a targeted effect on the estrogen binding site on the CYP2B1/2 and CYP1A genes apart from the PhB-responsive unit, or a second pathway (possibly sulfation) that is not PhB-inducible.

Environmental polychlorinated biphenyl exposure and cytochromes P450 in raccoons (Procyon lotor)

An investigation involving raccoons as a sentinel species at the Paducah Gaseous Diffusion Plant (PGDP) and Ballard Wildlife Management Area in western Kentucky (USA) delineated the extent of exposure to polychlorinated biphenyls (PCBs). Three separate measures of hepatic cytochrome P450 (CYP) induction were used to evaluate raccoon physiological responses to PCB exposure. Hepatic CYP induction was estimated via determination of total CYP, dealkylase activities, and immunoreactive proteins. There were no differences in raccoon biomarker responses between study sites. Significant relationships between and among PCB residues and biomarkers indicated that hepatic CYP induction had occurred in response to PCB exposure. Pentoxyresorufin O-deethylase (PROD) activity, CYP1A1, and CYP1A2 were biomarkers most closely associated with PCB exposure. The rank order of responses was CYP1A1 > CYP1A2 > PROD > ethoxyresorufin O-deethylase (EROD) as related to raccoon liver PCB concentrations, whereas the order was CYP1A1 > PROD > EROD > CYP1A2 when regressed with total PCB concentrations in abdominal fat.

Ecotoxicological Risks Associated with Land Treatment of Petrochemical Wastes. II. Effects on Hepatic Phase I and Phase II Detoxification Enzymes in Cotton Rats

The purpose of this study was to evaluate possible exposure and resultant hepatic effects of petrochemical waste on cotton rats (Sigmodon hispidus) living on landfarmed sites. Male and female cotton rats were collected in summer, fall, and winter from four landfarm sites and four ecologically similar reference sites. Hepatic methoxyresorufin O-deethylase (MROD) activity was significantly induced in male and female rats collected from landfarms compared to rats collected from reference sites. In contrast, changes in ethoxyresorufin O-deethylase (EROD) activity were inconsistent due to season, sex, and treatment variation. A significant decrease in EROD and MROD activity was found in cotton rats held for 48 h prior to sacrifice compared to rats euthanized on the day of capture. These results indicate that when using hepatic EROD and MROD activities as biochemical markers of exposure to aryl hydrocarbon receptor agonists, animals should be euthanized as quickly as possible after capture. The cotton rats collected from one landfarm unit exhibited a pattern of consistent elevation of EROD, MROD, and pentoxyresorufin O-deethylase (PROD) activity. This unit also had a pattern of elevated CYP1A2 protein expression determined by Western blotting. There were no consistent alterations from contaminant exposure on hepatic glutathione S-transferase (GST) activity, glutathione levels, or CYP1A1 protein. Hepatic EROD and MROD activities varied considerably between seasons and sex of rats. In conclusion, consistent induction of hepatic EROD and MROD activities in cotton rats was found in three out of four sampled landfarm sites compared to the rats collected from the reference sites, indicating exposure to contaminants--likely polyaromatic hydrocarbons.

Differential Effects of Two Lots of Aroclor 1254 on Enzyme Induction, Thyroid Hormones, and Oxidative Stress

Aroclor 1254 is a commercial mixture of polychlorinated biphenyls (PCBs), which is defined as being 54% chlorine by weight. However, the congener composition varies from lot to lot. Two lots which have been used in toxicity studies, 124-191 and 6024 (AccuStandard), were analyzed for their congener composition. Lot 6024 has approximately 10 times the dioxin toxic equivalents (TEQ) of lot 124-191. The purpose of this study was to determine if the difference in the TEQ of the two lots explains the different in vivo responses seen on a weight basis. Male Long-Evans rats (70 days old) were treated orally with a single dose of 0-1,000 mg/kg of each lot. Hepatic ethoxy-, methoxy-, and pentoxyresorufin O-deethylase (EROD, MROD, and PROD, respectively) activities as well as serum thyroxine (T(4)) concentrations and measures of oxidative stress were determined 4 days after treatment. Results, on a weight basis, indicate that lot 6024 led to a greater induction of EROD, MROD, and PROD but not total T(4) reduction. The differences in TEQ between the lots explained the differential induction of EROD and MROD but did not account for the induction of PROD nor decreases in T(4). PROD induction is not due to dioxin-like congeners, whereas the decrease in serum T(4) levels may involve multiple mechanisms. Effects on the antioxidants ascorbic acid and uric acid were seen only at the highest mass dose for both lots and were not explained by the difference in TEQ. These results illustrate that the differences in the TEQ explain the differences in the strict dioxin-like effects (EROD, MROD induction), but the non-dioxin-like congeners cause other effects that are not associated with the aryl hydrocarbon receptor (e.g., PROD). In addition, supra-additive effects also occur in the mixture (T(4), oxidative stress). Thus, current results demonstrate that overall toxicity cannot be predicted on the basis of the TEQ values. It is also critical that the lot number is reported in studies conducted with Aroclor 1254 because the congener composition and therefore the effects observed can be very different.