Induced cytochrome P-450 in intestine and liver of spot (Leiostomus xanthurus) from a polycyclic aromatic hydrocarbon contaminated environment

doi:10.1016/0166-445X(90)90026-L

Aquatic Toxicology

Volume 17, Issue 2, August 1990, Pages 119-131

https://doi.org/10.1016/0166-445X(90)90026-L Get rights and content

Abstract

Levels of total cytochrome P-450, of specific P-450 (determined immunologically with MAb 1-12-3 and referred to as P-450E) and ethoxyresorufin O-deethylase (EROD) were elevated in intestine and liver microsomes of spot (Leiostomus xanthurus) collected from the Elizabeth River, a polycyclic aromatic hydrocarbon (PAH) contaminated tributary of Chesapeake Bay. Fish were collected over a sediment PAH concentration gradient that ranged from 9 to 96 000 μg PAH/kg dry sediment. Intestinal P-450E was near the lower limits of detection in fish collected at the relatively clean sites but was elevated 80- to 100-fold in fish collected from contaminated sites. Intestinal EROD activity exhibited a similar trend. Liver P-450E and associated EROD activity was detectable in all samples and was induced approximately eight-fold at the most heavily contaminated site. Despite the sensitivity of the intestine to PAH inducing agents, intestinal P-450E levels did not correlate well with sediment PAH, whereas liver P-450E did. Instead, the intestinal enzyme was induced to similar and high levels at all contaminated sites. The results suggest that the intestine plays an important role in the absorption and metabolism of dietary PAH and/or other PAH-type inducing agents and that intestinal P-450E may be a useful indicator of exposure to these compounds via the diet.

References (33)

M.M. Bradford
A rapid and sensitive method for quantitation of microgram quantities of protein using the principle of protein-dye binding
Anal. Biochem.
(1976)
W.N. Burnette
‘Western blotting’, Electrophoretic transfer of proteins from sodium dodecyl sulfate —polyacrylamide gels to unmodified nitrocellulose and radiometric detection with antibody and radioiodinated protein A
Anal. Biochem.
(1981)
J.R. Foster et al.
Immunocytochemical localization of cytochrome P-450 in hepatic and extrahepatic tissues of the rat with monoclonal antibody against cytochrome P-450c
Biochem. Pharmacol.
(1986)
H.I. Friedman et al.
Intestinal fat digestion, absorption and transport
Am. J. Clin. Nutr.
(1980)
A. Goksøyr
Purification of hepatic microsomal cytochromes P-450 from β-naphthoflavone-treated Atlantic cod (Gadus morhua), a marine teleost fish
Biochim. Biophys. Acta
(1985)
R.J. Huggett et al.
Polynuclear aromatic hydrocarbons in the Elizabeth River, Virginia
A.V. Klotz et al.
An aryl hydrocarbon hydroxylating hepatic cytochrome P-450 from the marine fish (Stenotomus chrysops)
Arch. Biochem. Biophys.
(1983)
A.V. Klotz et al.
An alternative 7-ethoxyresorufin O-deethylase assay
D.C. Malins et al.
Neoplastic diseases in fish in relation to toxic chemicals: an overview
Aquat. Toxicol.
(1988)
M.R. Miller et al.
Cytochrome P-450 induction and localization in gill pillar (endothelial) cells of scup and rainbow trout
Aquat. Toxicol.
(1989)

P.A. Van Veld et al.

Dietary fat inhibits the intestinal metabolism of the carcinogen benzo[a]pyrene in fish

J. Lipid Res.

(1987)

P.A. Van Veld et al.

Effect of preexposure to dietary benzo[a]pyrene (BP) on the first-pass metabolism of BP by the intestine of toadfish (Opsanus tau): in vivo studies using portal vein-catheterized fish

Toxicol. Appl. Pharmacol.

(1988)

R.D. Vetter et al.

Coassimilation of dietary fat and benzo[a]pyrene in the small intestine

D.E. Williams et al.

Benzo[a]pyrene-hydroxylase catalyzed by purified isozymes of cytochrome P-450 from β-naphthoflavone-fed rainbow trout

Biochem. Pharmacol.

(1984)

P.C. Baumann

PAH, metabolites and neoplasia in feral fish populations

R.H. Bieri et al.

Polycyclic aromatic hydrocarbons in surface sediments from the Elizabeth River subestuary

Int. J. Environ. Anal. Chem.

(1986)

Cited by (107)

Biliary polycyclic aromatic hydrocarbons and enzymatic biomarkers in Eugerres brasilianus along four tropical estuaries
2021, Marine Pollution Bulletin
Citation Excerpt :
Induction of EROD verified in this study in association with the estimated bile PAHs in BJES and BPEC confirms the relevance of these parameters to monitor and diagnose estuarine aquatic ecosystems contaminated with PAHs (Gagnon and Rawson, 2017; Payne et al., 1987). Curiously, although aquatic sediment PAH contamination does not necessarily indicate internal accumulation of PAHs by local fish, strong correlations between sediment PAH contamination and EROD induction were found in spot Leiostomus xanthurus sampled along a sediment PAH concentration gradient in Elizabeth River, Chesapeake Bay estuary (Van Veld et al., 1990). The same trend was verified in winter flounder Pseudopleuronectes americanus along a gradient of increasing sediment PAH concentrations at Sydney Harbour estuary, Nova Scotia (Addison et al., 1994).
Polycyclic aromatic hydrocarbons (PAH) in bile and biochemical biomarkers were evaluated in Brazilian mojarra Eugerres brasilianus along four estuaries in northeastern Brazil. Bile PAHs naphthalene, phenanthrene chrysene, pyrene and benzo[a]pyrene were lowest at Formoso River Estuarine System (FRES), an area with low population density focused on tourism. Fish sampled in Suape Estuarine Complex (SEC), where a growing industrial port complex is established indicated higher naphthalene and pyrene concentrations compared with FRES. Fish sampled in highly urbanized and populated Bacia do Pina Estuarine Complex (BPEC) and Barra de Jangada Estuarine System (BJES) indicated an increase in all PAHs compared to FRES. Activities of phase 1 Ethoxyresorufin-O-deethylase, phase 2 glutathione-S-transferase and antioxidant defense catalase were induced up to 20, 2 and 2-fold in BJES and BPEC compared to FRES. This study confirms E. brasilianus as an important sentinel species, providing baseline information for these tropical estuaries with different degrees of anthropogenic pressure.
In vitro-in vivo extrapolation of hepatic and gastrointestinal biotransformation rates of hydrophobic chemicals in rainbow trout
2020, Aquatic Toxicology
Citation Excerpt :
These hydroxylation reactions are catalyzed predominantly by CYP1A, although other CYP enzymes may contribute (Schlenk et al., 2008). Observed differences in biotransformation of PYR and BAP in liver and intestinal S9 fractions (liver > intestine) were similar to tissue-specific differences in EROD activity shown here (Fig. 2A) and in previous studies (James et al., 1997; Van Veld et al., 1988, 1990; 1991). This finding is also consistent with relative differences in measured in vitro intrinsic clearance of BAP determined in liver and intestinal cells lines (Stadnicka-Michalak et al., 2018).
Hepatic in vitro biotransformation assays, in combination with in vitro-in vivo extrapolation (IVIVE) and bioaccumulation modeling, can be used to support regulatory bioaccumulation assessments. In most applications, however, these methods ignore the possibility of extrahepatic metabolism. Here we evaluated intestinal biotransformation in rainbow trout using S9 fractions prepared from the upper intestinal (GIT) epithelium. Measured levels of activity determined using standard substrates for phase I and phase II biotransformation enzymes were within 2-fold of activities measured in hepatic S9 fractions. In vitro intrinsic clearance rates for 2-ethylhexyl-4-methoxycinnamate (EHMC; an organic sunscreen agent) and two polycyclic aromatic hydrocarbons (pyrene [PYR] and benzo(a)pyrene [BAP]) were significantly higher in liver S9 fractions than in GIT S9 fractions. For octocrylene (OCT; a second sunscreen agent), however, in vitro intrinsic clearance rates were higher in GIT S9 fractions compared to liver S9 fractions. An existing ‘liver only’ IVIVE model was expanded to consider biotransformation in both the liver and GIT. Relevant IVIVE scaling factors were developed by morphological, histological, and biochemical evaluation of trout intestines. For chemicals biotransformed at higher rates by hepatic S9 fractions (i.e., BAP, PYR, EHMC), the ‘liver & GIT’ model yielded whole-body biotransformation rate constants (k_MET) that were within 1.2 to 1.4-fold of those estimated using the ‘liver only’ model. In contrast to these findings, the mean k_MET for OCT obtained using the ‘liver & GIT’ model was 3.3 times higher than the mean k_MET derived using the ‘liver only’ model and was in good agreement with empirical k_MET estimates determined previously for trout (<20 % difference). The results of this study suggest that current ‘liver only’ IVIVE approaches may underestimate in vivo biotransformation rates for chemicals that undergo substantial biotransformation in the GIT.
Effects of chronic dietary petroleum exposure on reproductive development in polar cod (Boreogadus saida)
2016, Aquatic Toxicology
Citation Excerpt :
The overall low amounts of PAHs in the exposure diet and the domination of lower weight PAHs with a potential inhibitory action on EROD activity may have limited the induction of responses at the doses used in the present study. Furthermore, inclusion of intestinal EROD activity to identify potential metabolism occurring prior to systemic uptake of the PAHs and alkylated PAHs would have provided additional information on the dietary exposure, especially at low levels (James et al., 1997; Van Veld et al., 1990). Such complementary analyses may allow the comparison of tissue-specific biotransformation capacity and has previously been found relevant for polar cod exposed to dietary crude oil (Nahrgang et al., 2010b).
Increasing human activities in the Arctic raise the risk of petroleum pollution, thus posing an elevated risk for Arctic organisms to be chronically exposed to petroleum compounds. The endocrine disrupting properties of some of these compounds (i.e. polycyclic aromatic hydrocarbons [PAHs]) present in crude oil may have negative effects on the long and energy intensive reproductive development of polar cod (Boreogadus saida), an Arctic keystone species. In the present study, selected reproductive parameters were examined in feral polar cod exposed to crude oil via a natural diet (0.11, 0.57 and 1.14 μg crude oil/g fish/day [corresponding to low, medium and high treatments, respectively]) for 31 weeks prior to spawning. Fish maturing in the current reproductive period made up 92% of the experimental population while 5% were immature and 3% were identified as resting fish. Phase I metabolism of PAHs, indicated by ethoxyresorufin-O-deethylase (EROD) activity, showed a dose-dependent increase in high and medium crude oil treatments at week 6 and 22, respectively. Decreasing EROD activity and increasing PAH bile metabolite concentrations over the experimental period may be explained by reproductive maturity stage. Significant alterations in sperm motility were observed in crude oil exposed males compared to the controls. The investigated somatic indices (gonad and hepatic), germ cell development and plasma steroid levels (estradiol-17β [females], testosterone [males and females] and 11-ketotestosterone [males]) were not significantly altered by chronic dietary exposure to crude oil. The environmentally realistic doses polar cod were chronically exposed to in this study were likely not high enough to induce adverse effects in this ecologically important fish species. This study elucidated many baseline aspects of polar cod reproductive physiology and emphasized the influence of maturation state on biomarkers of PAH biotransformation (EROD and PAH bile metabolites).
Towards science-based sediment quality standards-Effects of field-collected sediments in rainbow trout (Oncorhynchus mykiss)
2015, Aquatic Toxicology
Sediments can act as long-term sinks for environmental pollutants. Within the past decades, dioxin-like compounds (DLCs) such as polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), polychlorinated biphenyls (PCBs), and polycyclic aromatic hydrocarbons (PAHs) have attracted significant attention in the scientific community. To investigate the time- and concentration-dependent uptake of DLCs and PAHs in rainbow trout (Oncorhynchus mykiss) and their associated toxicological effects, we conducted exposure experiments using suspensions of three field-collected sediments from the rivers Rhine and Elbe, which were chosen to represent different contamination levels. Five serial dilutions of contaminated sediments were tested; these originated from the Prossen and Zollelbe sampling sites (both in the river Elbe, Germany) and from Ehrenbreitstein (Rhine, Germany), with lower levels of contamination. Fish were exposed to suspensions of these dilutions under semi-static conditions for 90 days. Analysis of muscle tissue by high resolution gas chromatography and mass spectrometry and of bile liquid by high-performance liquid chromatography showed that particle-bound PCDD/Fs, PCBs and PAHs were readily bioavailable from re-suspended sediments. Uptake of these contaminants and the associated toxicological effects in fish were largely proportional to their sediment concentrations. The changes in the investigated biomarkers closely reflected the different sediment contamination levels: cytochrome P450 1A mRNA expression and 7-ethoxyresorufin-O-deethylase activity in fish livers responded immediately and with high sensitivity, while increased frequencies of micronuclei and other nuclear aberrations, as well as histopathological and gross pathological lesions, were strong indicators of the potential long-term effects of re-suspension events.
Our study clearly demonstrates that sediment re-suspension can lead to accumulation of PCDD/Fs and PCBs in fish, resulting in potentially adverse toxicological effects. For a sound risk assessment within the implementation of the European Water Framework Directive and related legislation, we propose a strong emphasis on sediment-bound contaminants in the context of integrated river basin management plans.
Biomarkers and bioaccumulation of clam Ruditapes philippinarum in response to combined cadmium and benzo[α]pyrene exposure
2011, Food and Chemical Toxicology
Citation Excerpt :
Therefore, it is not difficult to explain why there was more Cd accumulated in clams exposed to mixtures contaminants than in single Cd exposure. For BaP metabolism, it has been established that once the BaP enter the organism’s system they can experience a series of oxidative processes mediated by the cytochrome P450 system (phase I) which can be assessed by measuring ethoxyresorufin-O-deethylase (EROD) and AHH activities (Collier and Varanasi, 1991; Van Veld et al., 1990). Then the phase I by-products may undergo further metabolism by conjugative enzymes (phase II) such as GST, which will make them more readily excretable.
Biochemical and molecular biomarkers (the contents of metallothionein (MT), glutathione (GSH), the activities of aryl hydrocarbon hydroxylase (AHH), glutathione S-transferase (GST) and superoxide dismutase (SOD) and the mRNA expressions of GST-pi and Cu, Zn-SOD) were evaluated in clams Ruditapes philippinarum exposed to cadmium (Cd, 15 μg/L) and benzo[α]pyrene (BaP, 0.01 μg/L) individually and in combination (15 μg/L Cd + 0.01 μg/L BaP) for 21 days. The accumulation of Cd, BaP and the biomarkers measured in the gills and digestive glands of the clam showed significant increase in combination treatment and it was significantly higher than the Cd or BaP treatment (P > 0.05). The contents of MT increased in Cd and Cd + BaP treatment, while AHH activities were increased in Bap and Cd + BaP treatment (P > 0.05). GSH levels enhanced in Cd group and declined significantly in Cd + BaP treatment (P > 0.05). The activities of GST, SOD, and mRNA expressions of GST-pi, Cu, Zn-SOD increased remarkably in the clams exposed to combined pollutants. In this study, a significant interaction was observed for Cd and BaP accumulation in the clam and the current findings demonstrate the differences in antioxidant response of the biomarkers in clam to single contaminant and the mixtures.
Up-regulation of hepatic ABCC2, ABCG2, CYP1A1 and GST in multixenobiotic-resistant killifish (Fundulus heteroclitus) from the Sydney Tar Ponds, Nova Scotia, Canada
2009, Marine Environmental Research
Cellular defence against accumulation of toxic xenobiotics includes metabolism by phase I and II enzymes and export of toxicants and their metabolites via ATP-binding cassette (ABC) transporters. Liver gene expression of representatives of these three protein groups was examined in a population of multixenobiotic-resistant killifish (Fundulus heteroclitus) from the Sydney Tar Ponds, Nova Scotia, Canada. The Tar Ponds are heavily polluted with polycyclic aromatic hydrocarbons, polychlorinated biphenyls and heavy metals. The relationship among ABC transporters ABCB1, ABCB11, ABCC2, ABCG2, phase I enzyme cytochrome P4501A1 (CYP1A1) and phase II enzyme glutathione-S-transferase (GST-mu) was investigated by quantifying hepatic transcript abundance. In Tar Pond killifish, hepatic mRNA expression levels of ABCC2, ABCG2, CYP1A1 and GST-mu were elevated compared to reference sites, suggesting that hydrophobic contaminants undergo phase I and II metabolism and are then excreted into the bile of these fish. Hepatic ABCB1 and ABCB11 mRNA were not up-regulated in Tar Pond fish compared to two reference sites, indicating that these two proteins are not involved in conferring multixenobiotic resistance to Tar Pond killifish. The results suggest instead that liver up-regulation of phase I and II enzymes and complementary ABC transporters ABCC2 and ABCG2 may confer contaminant resistance to Tar Pond fish.

View all citing articles on Scopus

View full text

Article preview

Aquatic Toxicology

Abstract

References (33)

A rapid and sensitive method for quantitation of microgram quantities of protein using the principle of protein-dye binding

Anal. Biochem.

‘Western blotting’, Electrophoretic transfer of proteins from sodium dodecyl sulfate —polyacrylamide gels to unmodified nitrocellulose and radiometric detection with antibody and radioiodinated protein A

Anal. Biochem.

Immunocytochemical localization of cytochrome P-450 in hepatic and extrahepatic tissues of the rat with monoclonal antibody against cytochrome P-450c

Biochem. Pharmacol.

Intestinal fat digestion, absorption and transport

Am. J. Clin. Nutr.

Purification of hepatic microsomal cytochromes P-450 from β-naphthoflavone-treated Atlantic cod (Gadus morhua), a marine teleost fish

Biochim. Biophys. Acta

Polynuclear aromatic hydrocarbons in the Elizabeth River, Virginia

An aryl hydrocarbon hydroxylating hepatic cytochrome P-450 from the marine fish (Stenotomus chrysops)

Arch. Biochem. Biophys.

An alternative 7-ethoxyresorufin O-deethylase assay

Neoplastic diseases in fish in relation to toxic chemicals: an overview

Aquat. Toxicol.

Cytochrome P-450 induction and localization in gill pillar (endothelial) cells of scup and rainbow trout

Aquat. Toxicol.

Dietary fat inhibits the intestinal metabolism of the carcinogen benzo[a]pyrene in fish

J. Lipid Res.

Effect of preexposure to dietary benzo[a]pyrene (BP) on the first-pass metabolism of BP by the intestine of toadfish (Opsanus tau): in vivo studies using portal vein-catheterized fish

Toxicol. Appl. Pharmacol.

Coassimilation of dietary fat and benzo[a]pyrene in the small intestine

Benzo[a]pyrene-hydroxylase catalyzed by purified isozymes of cytochrome P-450 from β-naphthoflavone-fed rainbow trout

Biochem. Pharmacol.

PAH, metabolites and neoplasia in feral fish populations

Polycyclic aromatic hydrocarbons in surface sediments from the Elizabeth River subestuary

Int. J. Environ. Anal. Chem.

Cited by (107)

Biliary polycyclic aromatic hydrocarbons and enzymatic biomarkers in Eugerres brasilianus along four tropical estuaries

In vitro-in vivo extrapolation of hepatic and gastrointestinal biotransformation rates of hydrophobic chemicals in rainbow trout

Effects of chronic dietary petroleum exposure on reproductive development in polar cod (Boreogadus saida)

Towards science-based sediment quality standards-Effects of field-collected sediments in rainbow trout (Oncorhynchus mykiss)

Biomarkers and bioaccumulation of clam Ruditapes philippinarum in response to combined cadmium and benzo[α]pyrene exposure

Up-regulation of hepatic ABCC2, ABCG2, CYP1A1 and GST in multixenobiotic-resistant killifish (Fundulus heteroclitus) from the Sydney Tar Ponds, Nova Scotia, Canada