Integrated approach for environmental assessment of new and existing substances.

To ensure the environmental safety of new and existing substances, the environmental fate and potential effects resulting from their release into the environment must be assessed. This requires the development of reasonable, consistent, and effective methods to conduct environmental risk assessments and to prioritize testing for these substances. This assessment must integrate fate and effects at the point-of-entry; it should also extend to an assessment of the potential to persist, and the consequences of increases in exposure concentrations, and to bioaccumulate. The conventional environmental risk assessment approach is used to assess the fate and effects of a substance at its point-of-entry into the environment. In this paper, an approach is presented for conducting quantitative environmental risk assessments of new and existing substances that builds on this conventional approach by including quantitative assessment of the potential for, and consequences of, persistence and bioaccumulation. The approach is described for aquatic, sediment, and terrestrial environments. For each environmental compartment, the approach includes (i) classification of the substance, based on environmental partitioning processes, to ensure that the appropriate data are collected and models used; (ii) a fate assessment to understand the ultimate fate of the substance after entry into the environment or "an environmental compartment" and to predict the exposure concentration of the substance at point-of-entry; (iii) a persistence assessment which determines the potential for increase in the exposure concentration as a result of repeated additions of the substance; (iv) effects assessment; (v) environmental risk assessment to examine the potential for adverse impact on ecosystems; and (vi) a bioaccumulation assessment to evaluate the potential for direct and indirect effects on the species of interest due to bioaccumulation. The assessment approach is illustrated using data for a hypothetical consumer product substance that is disposed down-the-drain.

Surfactant bioconcentration--a critical review.

Bioconcentration data for surfactants have been collected and critically reviewed. Twenty-two references report whole body bioconcentration data. Most of these data are inappropriate to quantitatively describe the bioconcentration of surfactants because the most frequently used analytical method, LSC without prior chromatographic separation of radiolabelled compounds, does not allow to distinguish between parent compound and metabolites. Hence, the measured concentrations very likely are overestimates of the concentration of the parent surfactant. In order to compare data we defined a comparability criterion. Data which fulfil this criterion consistently overestimate the true extent of bioconcentration. Fifty-four out of 100 whole body concentration ratios (CRs) were selected employing the above criterion, with 33, 11, and 10 Crs reported for anionic, cationic and nonionic surfactants, respectively. Further findings are: 1. Selected CRs range between 2.4 for octyltrimethylammonium chloride and 1960 for tallowtrimethylammonium chloride. In general, CRs increased with increasing alkyl chain length. 2. Surfactants of all classes are readily taken up across the gills. Hexadecylpyridinium and dialkyl dimethyl ammonium surfactants appear to be taken up rather slow. 3. Nonionic and anionic surfactants were demonstrated to be biotransformed. Tissue specific data demonstrated that elimination via the gall bladder is an important excretion route. 4. Environmental variables appeared to influence bioconcentration of ionic surfactants.

Turnover of hepatic microsomal cytochrome P4501A protein and heme in beta-naphthoflavone-induced Fundulus heteroclitus.

Earlier studies showed that in Fundulus heteroclitus liver, the content of microsomal cytochrome P4501A (CYP1A) protein induced by beta-naphthoflavone is elevated for weeks after its induction, whereas the content of the induced CYP1A mRNA peaks and declines rapidly (2-4 days). This finding suggests an unusually long half-life for teleost CYP1A. We examined directly the half-live of hepatic microsomal CYP1A protein and heme moieties in F. heteroclitus, by measuring the incorporation and disposition of [3H]-leucine and [14C]-5-aminolevulinic acid (ALA) in fish that had been treated with the CYP1A inducer beta-naphthoflavone prior to administration of label. Incorporation of [3H]-leucine into trichlororacetic acid-precipitable (total) microsomal protein and into immunoprecipitated CYP1A protein peaked between 1.5 to 4 hours. Incorporation of [14C]-ALA into total microsomal (heme) protein peaked at 24 hours, and into the heme of CYP1A at 8 hours. Loss of label from total microsomal protein was biphasic, giving half-lives of 8 and 138 hours for bulk protein, and 66 and 141 hours for heme. CYP1A apoprotein had a half-life of 32 to 39 hours based on isotopic loss and 43 hours calculated from the kinetics of enzyme induction. The apparent half-life of CYP1A heme was approximately 100 hours, which was substantially greater than the protein half-life, in contrast to mammalian P450s, in which half-lives for protein and heme are similar. The distinction in heme half-lives could represent fundamental differences between fish and mammals in P450 heme/protein interactions. The protein half-life is inconsistent with a prolonged stability of CYP1A, yet 30% of the [3H] and 40% of the [14C] seen in CYP1A at the peak of incorporation could still be measured after eight days. Mechanisms other than inherently long-lived protein, perhaps stabilization or enhanced translation of a minor mRNA pool, might contribute to persistence of CYP1A.

Effects of temperature acclimation on the expression of hepatic cytochrome P4501A mRNA and protein in the fish Fundulus heteroclitus.

Previous studies showed that hydrocarbon induction of hepatic microsomal monooxygenase activity is attenuated in the teleost fish Fundulus heteroclitus acclimated to low temperature. The basis of that attenuation, and the effects of temperature on monooxygenase activity, were examined by analyzing liver cytochrome P4501A (CYP1A) mRNA, protein, and catalytic activity in control and beta-naphthoflavone (BNF)-treated F. heteroclitus acclimated to 6 or 16 degrees C. There were no temperature-related differences in total P450 content, NADPH-cytochrome c (P450) reductase activity, ethoxyresorufin O-deethylase (EROD) activity, or immunoquantified CYP1A content in hepatic microsomes of untreated fish. Fish acclimated to 16 degrees C and given a single intraperitoneal injection of BNF exhibited a rapid rise and fall in CYP1A mRNA content and an induction of EROD activity and CYP1A protein that was undiminished over 7 days. Similarly treated fish acclimated at 6 degrees C showed an increase in CYP1A mRNA content greater than that in 16 degrees C fish, but with no significant increase in EROD activity or CYP1A content over 7 days. Examined over a longer term, microsomal EROD activity was significantly induced by BNF in fish at both temperatures; activity peaked at 5-7 days in 16 degrees C fish, while in 6 degrees C fish the activity continued to rise slowly over 25 days. However, the greatest activity reached in 6 degrees C fish (0.68 nmol/min/mg) was less than half that seen in the warmer animals (1.46 nmol/min/mg). Immunodetectable CYP1A content showed the same trend as EROD activity, and the turnover number (nmol product formed/min/nmol CYP1A) for EROD activity was about the same in all groups, indicating that concentration of the catalyst alone could account for the different patterns of microsomal activity. CYP1A mRNA content was again induced to a similar degree by BNF in both the 6 and the 16 degrees C fish; the apparent half-life of the mRNA was substantially longer in cold-acclimated than in warm-acclimated BNF-treated fish. Comparing the levels of CYP1A mRNA and protein at the two acclimation temperatures following BNF treatment indicates that translational activity, rather than transcriptional activity, is the sensitive point in the effect of temperature on CYP1A induction in these fish.

Effects of ortho- and non-ortho-substituted polychlorinated biphenyl congeners on the hepatic monooxygenase system in scup (Stenotomus chrysops).

Polychlorinated biphenyl congeners that are abundant in environmental samples, and known to induce hepatic monooxygenase isozymes in the P450IA gene subfamily in mammals, were examined for their ability to induce hepatic monooxygenase activity in scup, a marine teleost. Scup were dosed ip with 3,3',4,4'-tetrachlorobiphenyl (congener 77), 2,3,3',4,4'-pentachlorobiphenyl (congener 105), 2,3',4,4',5-pentachlorobiphenyl (congener 118), 2,2',3,4,4',5'-hexachlorobiphenyl (congener 138), 2,2',3,3',4,4'-hexachlorobiphenyl (congener 128), or beta-naphthoflavone and examined for increases in ethoxyresorufin O-deethylase (EROD) activity, immunodetectable cytochrome P450E (the EROD catalyst in scup), and in vitro translatable mRNA for P450E. Monooxygenase parameters were significantly induced only by 3,3',4,4'-tetrachlorobiphenyl (TCB). However, while translatable mRNA for P450E was induced at all doses (1, 5, and 10 mg/kg), EROD activity and P450E were decreased at the 5 and 10 mg/kg doses, relative to the response at 1 mg/kg. A strong relationship between residual TCB concentration in the liver and the decreased EROD activity was evident at the higher doses of TCB. Aminopyrine N-demethylase, a monooxygenase activity not catalyzed by P450E, was unaffected by TCB treatment, indicating a specificity in the TCB effect. Analysis in vitro revealed that TCB was a potent competitive inhibitor of EROD activity, with half-maximal inhibition at 0.3 microM, near the Km for ethoxyresorufin, suggesting one mechanism for the in vivo effect of TCB. These results demonstrate that PCB congeners with ortho-chlorine substitution, and which are effective inducers of AHH and EROD activity in mammals, are ineffective, at the doses tested, as inducers in the teleost scup.

The temporal relationships between P450E protein content, catalytic activity, and mRNA levels in the teleost Fundulus heteroclitus following treatment with beta-naphthoflavone.

P450 induction occurs in some marine organisms following chemical exposure. The mode of 3-methylcholanthrene (MC)-type induction was evaluated by examining hepatic isozyme P450E content, catalytic activity, and mRNA levels in the marine teleost Fundulus heteroclitus after exposure to a single dose of beta-naphthoflavone (BNF). P450E is the major teleost P450 induced by MC-type compounds and is the catalyst for aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) activities. In a 20-day experiment, EROD activity was elevated in BNF-treated animals from Day 4 through Day 20. Increases in immunodetectable P450E showed the same trend as EROD activity, with consistently low control values and at least a 19-fold increase in the BNF-treated fish. Precipitation of liver RNA in vitro translation products with anti-P450E antibody gave no detectable signal from control fish, while the BNF-treated animals showed incorporation of [3H]leucine in a single 6,000 Mr band. In a shorter term experiment, EROD activity and P450E levels were again coordinately increased in response to BNF treatment, and immunoprecipitation of translation products from these fish showed a clear trend of increased P450E mRNA levels for all time points 6 h or more post-treatment. Hybridization of RNA from BNF-treated Fundulus with a trout P450IA1 cDNA also showed increases in a single band with time. The increases in P450E mRNA preceded increases in P450E protein and enzyme activity by about 25 h. However, P450E mRNA declined rapidly, reaching control levels by 5 days, while protein levels remained elevated for at least 13 days. The results support a hypothesis that transcriptional enhancement is involved in induction of MC-inducible P450s in fish, but indicate that P450E induction is also under other forms of regulatory control.

Cytochrome P-450 isozymes and monooxygenase activity in aquatic animals.

The roles of different forms of cytochrome P-450 in activation and deactivation of toxic chemicals, synthesis and breakdown of steroid hormones, and other functions, indicate the significance of these enzymes. Monooxygenase systems have been studied in species from several phyla of aquatic organisms. However, cytochrome P-450, the dominant catalyst in xenobiotic monooxygenase activity, is best studied in fish. Forms of cytochrome P-450 have been purified from several teleost species, including scup (Stenotomus chrysops), rainbow trout (Salmo gairdneri), and cod (Gadus morhua). Cytochrome P-450E from scup, cytochrome P-450 LM4b from trout, and cytochrome P-450c from cod have properties similar to each other and appear to be homologous hydrocarbon or BNF-inducible isozymes. Partially purified cytochrome DBA-P-450-I from little skate, Raja erinacea, is possibly an elasmobranch counterpart of these teleost forms. Cytochrome P-450E from scup is immunochemically related to the major BNF-inducible isozyme (cytochrome P-450c or BNF-B) in rats, indicating homology between the fish and mammalian BNF-inducible isozymes. Several other cytochrome P-450 forms with interesting or unusual properties have been purified from aquatic species. Mammalian homologs are not yet known for these isozymes. Further studies of cytochrome P-450 forms in aquatic species should establish additional homologies and the regulation of these forms by chemical and biological variables, possibly providing fundamental insights into the function and evolution of these proteins.

Specificity and cross-reactivity of monoclonal and polyclonal antibodies against cytochrome P-450E of the marine fish scup.

Monoclonal antibody (MAb) 1-12-3 generated against liver cytochrome P-450E (P-450E), an aryl hydrocarbon hydroxylase of the marine fish Stenotomus chrysops (scup), reacted only with P-450E when tested in immunoblot analysis with five P-450 fractions from scup liver. This and six other MAbs against P-450E recognized purified P-450E, as well as a single band in beta-naphthoflavone (BNF)-induced scup microsomes that comigrated with authentic P-450E. Like MAb 1-12-3, polyclonal anti-P-450E reacted with P-450E but not with other scup P-450 fractions and reacted strongly with a band coincident to P-450E in BNF-treated scup microsomes. However, the polyclonal antibody (PAb) also faintly recognized additional microsomal proteins. MAb 1-12-3 recognized P-450E induced by 3,3',4,4',5,5'-hexachlorobiphenyl and by polychlorinated biphenyl mixtures in scup, and a single band induced by BNF or 3-methylcholanthrene (MC) in microsomes of other teleosts, including two trout species, killifish and winter flounder. The content of the P-450E counterpart in these fish and also in untreated scup coincided with induced ethoxyresorufin O-deethylase (EROD) activity. Induced EROD activity in scup and trout was strongly inhibited by MAb 1-12-3, further demonstrating the relationship between P-450E and induced P-450E in trout. MAb 1-12-3, two other MAbs, and anti-P-450E PAb recognized a band comigrating with P-450c in BNF-induced rat microsomes. MAb 1-12-3 also recognized purified rat P-450c. MAb 1-12-3 and anti-P-450E PAb recognized a second band of lower molecular weight than P-450c in BNF rat microsomes which may correspond to P-450d, the MC- and isosafrole-inducible rat isozyme. The results firmly establish the identity of scup P-450E, the relationship of BNF-induced P-450 in other teleosts with P-450E, and the immunochemical relationship of P-450E with rat P-450c. Furthermore, results with untreated fish suggest that effects of environmental chemicals may be detected by immunoblotting with monoclonal anti-P-450E.

Monoclonal antibodies to liver microsomal cytochrome P-450E of the marine fish Stenotomus chrysops (scup): cross reactivity with 3-methylcholanthrene induced rat cytochrome P-450.

Hybridomas were prepared from myeloma cells and spleen cells of BALB/c female mice immunized with hepatic cytochrome P-450E purified from the marine fish, Stenotomus chrysops (scup). Nine independent hybrid clones produced MAbs, either IgG1, IgG2b, or IgM, that bound to purified cytochrome P-450E in radioimmunoassay. Antibodies from one clone MAb (1-12-3), also strongly recognized rat cytochrome P-450MC-B (P-450BNF-B; P-450c). The nine antibodies inhibited reconstituted aryl hydrocarbon hydroxylase (AHH) and ethoxycoumarin O-deethylase of scup cytochrome P-450E to varying degrees, and inhibited AHH activity of beta-naphthoflavone-induced scup liver microsomes in a pattern similar to that in reconstitutions, indicating that cytochrome P-450E is identical to the AHH catalyst induced in this fish by beta-naphthoflavone. MAb 1-12-3 also inhibited the reconstituted AHH activity of the major BNF-induced rat isozyme. Conversely, MAb 1-7-1 to rat cytochrome P-450MC-B had little effect on AHH activity of scup cytochrome P-450E, and did not recognize cytochrome P-450E in radioimmunoassay nor in an immunoblot. Scup cytochrome P-450E and rat cytochrome P-450MC-B thus have at least one common epitope recognized by MAb 1-12-3, but the epitope recognized by Mab 1-7-1 is absent or recognized with low affinity in cytochrome P-450E. The various assays indicate that the nine MAbs against cytochrome P-450E are directed to different epitopes of the molecule. These MAbs should be useful in determining phylogenetic relationships of the BNF- or MC-inducible isozymes and their regulation by other environmental factors.

Monooxygenase Induction and Chlorobiphenyls in the Deep-Sea Fish Coryphaenoides armatus.

Inhibition of liver microsomal ethoxyresorufin O-deethylase and aryl hydrocarbon hydroxylase activities by alpha-naphthoflavone and by polyclonal antibodies to hydrocarbon-inducible cytochrome P-450E from teleost liver indicated a xenobiotic-induced origin of these activities in the deep-sea fish Coryphaenoides armatus. Specific recognition of a protein by antibodies to P-450E in an immunoblot assay further indicated xenobiotic-induced cytochrome P-450 in these animals. Levels of apparently induced cytochrome P-450 and monooxygenase activity correlated positively with the tissue content of chlorobiphenyls of known inducing activity, implicating such compounds in biochemical effects occurring in the deep ocean.