Hepatic AHH activity

Simultaneous exposure of rats to Aroclor 1254 or 1260 and chemicals of environmental concern such as the pesticides mirex, photomirex, and/or kepone in the diet resulted in increased severity of the liver lesions attributed to exposure to chlorinated biphenyls alone (Chu et al. 1980). Induction of hepatic AHH activity by Aroclor 1254 in the diet of lactating rats was increased in an additive manner by simultaneous dietary exposure to polybrominated biphenyls such as Firemaster BP-6 (McCormack et al. 1979). 

It can also be concluded that dermal absorption occurred as evidenced by the development of skin tumors (Boutwell and Bosch 1958 Lijinsky et al. 1957 Poel and Kammer 1957 Roe et al. 1958) and lung tumors (Roe et al. 1958) in mice following the dermal application of coal tar creosote (see Section 3.2.3.7). Other studies in animals support absorption of coal tar products after dermal application. Coal tar solution (0.05 mL of a 20% solution) was applied to the skin of six neonatal rats (4-6 days of age) and 24 hours later AHH activity was measured in the skin and liver (Bickers and Kappas 1978). There was greater than a 10-fold induction of skin AHH activity (298 13 versus 26.3 19 pmol hydroxy-benzo[a]pyrene/mg protein/hour in controls) and marked increased hepatic AHH activity (16,300 899 versus 750 35 pmol hydroxy-benzo[a]pyrene/mg protein/hour in controls) after topical application of the coal tar solution. 

Coal tar solution (0.05 mL of a 20% solution) was applied to the skin of six neonatal rats (4-6 days of age), and 24 hours later, AHH activity was measured in the skin and liver (Bickers and Kappas 1978). There was greater than a 10-fold induction of skin AHH activity (298 13 versus 26.3 19 pmol hydroxy benzopyrene/mg protein/hour in controls) and marked increased hepatic AHH activity... 

In this report we compare several properties of hepatic microsomal AHH activity in control and DBA-treated little skates (including metabolic profiles obtained from c-benzo(a)pyrene as elucidated by high pressure liquid chromatography [HPLC]), we describe the partial purification of two different forms of cytochrome P-450 (cytochrome P-448 and cytochrome P-451) from hepatic microsomes of DBA-pretreated little skates and we report polycyclic hydrocarbon-like induction in large numbers of winter flounder assayed in Maine during June, July, and August, which was quite different than data obtained with sheepshead examined in Florida during the same period. 

Several properties of hepatic microsomal AHH activity were compared in control and DBA-pretreated male little skates as shown in Table I. Following treatment there was an approximately 35-fold increase in specific enzyme activity, as quantitated by fluorescence of the phenolic metabolites formed (3, 21). The pH optimum, which was fairly broad, and the concentration of benzo(a)-pyrene (0.06 mM) that had to be added to the incubation mixture to achieve maximum enzyme activity were the same for both control and induced skate hepatic microsomes. The shorter periods observed for linearity of product formation with microsomes from the induced skates is thought to be related to the much higher AHH activity present, and may be due to substrate depletion or the formation of products which are inhibitory (i.e., compete with the MFO system as they are substrates themselves). A similar explanation may be relevant for the loss of linear product formation at lower microsomal protein concentrations in the induced animals. 

The apparent kinetic constants were obtained from Lineweaver-Burk plots of AHH activities recorded in the presence of increasing concentrations of benzo(a)pyrene (0.001-1.0 mM). The plots were linear for both untreated and DBA-induced animals. The apparent V was 20- to 30-fold higher in hepatic microsomes from the induced skates whereas the apparent K values were of the same magnitude in control and treated fish. 

An obvious difference was also noted between control and induced skate hepaticdnicrosomal AHH activity in the presence of a-naphthoflavone (10 M). This compound, when added in vitro at this or higher concentrations, caused significant stimulation of AHH activity in control animals (about 3-fold) but inhibition (80%) was found in DBA-pretreated skates. Similar results were earlier reported for control and 3-methylcholanthrene-treated rats (23), where it appears that the response is due to differential effects of a-naphthoflavone on hepatic microsomal cytochrome P-450 (stimulated) and cytochrome P-448 (inhibited) (24). Our data suggests that there may be a novel form of cytochrome P-450 synthesized in skate liver in response to polycyclic hydrocarbon administration, even though there was no hypsochromic shift in the carbon monoxide difference spectrum of dithionite reduced hepatic microsomes from DBA-treated skates (relative to hepatic microsomes from control fish). 

SOME PROPERTIES OF HEPATIC MICROSOMAL BENZ0(a)PYRENE HYDROXYLASE (AHH) ACTIVITY IN CONTROL AND... 

Collectively, these experiments have demonstrated that substantial amounts of cytochrome P-448 are indeed present in liver of DBA-pretreated male and female skates but not in untreated skates. Sufficient cytochrome P-448 is present to account for the increased AHH activities observed in hepatic microsomes of DBA-treated skates. 

One of our more interesting observations is illustrated in Table III. The administration of DBA to winter flounder increased hepatic microsomal AHH and 7-ethoxyresorufin deethylase activities as expected, and AHH activity was strongly inhibited in the DBA-treated flounder by 10" M a-naphthoflavone as we have previously reported for both little skate (4) and sheepshead (9). However, the presence of high AHH and 7-ethoxyresorufin deethylase activities in one control flounder, and the inhibition of AHH activity by a-naphthoflavone in this animal suggested that the hepatic microsomal MFO system of this fish was already induced. 

Bend, J. R., Hall, P., and Foureman, G. L. Comparison of benzo(a)pyrene hydroxylase (aryl hydrocarbon hydroxylase, AHH) activities in hepatic microsomes from untreated and 1,2,3,4-dibenzanthracene (DBA)-induced little skate (Raja erinacea). Bull. Mt. Desert Island Biol. Lab. (1976) 16. 3-5. 

PCNs, particularly penta- and hexaCNs, induce hepatic ethoxyresorufin O-deethylase (EROD) and aryl hydrocarbon hydroxylase (AHH) activities [45,46] and oxidative stress resulting in increased lipid peroxidation, decreased hepatic vitamins A and E, and decreased catalase and superoxide... 

EFFECT OF 1,2,3,4-DIBENZANTHRACENE (DBA)-PRETREATMENT ON HEPATIC MICROSOMAL 3ENZ0(a)PYRENE HYDROXYLASE (AHH) AND 7-ETH0XYRES0RUFIN DEETHYLASE (7-ERF) ACTIVITIES IN WINTER FLOUNDER... 

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