James S. Felton

Species differences in the substrate specificity of hepatic cytochrome P-448 From polycyclic hydrocarbon-treated animals☆

Abstract The in vitro effects of α-naphthoflavone on four hepatic mono-oxygenase activities associated with aromatic hydrocarbon responsiveness in the mouse (aryl hydrocarbon hydroxylase, 2-acetylamino-fluorene N -hydroxylase, biphenyl 2-hydroxylase, and biphenyl 4-hydroxylase) were investigated before and after methylcholanthrene treatment of C57BL/6N and DBA/2N mice, rats, hamsters, guinea pigs and rabbits. The electrophoretic pattern of cytochrome P-450 subunits and reduced CO-hemoprotein difference spectra of the microsomal fractions were also studied. Pretreatment of animals with methylcholanthrene caused: (1) a 1.5 to 2 mm hypsochromic shift in the Soret peak of the reduced hemoprotein-CO complexes in liver microsomes from a C57BL/6N mouse, rat, hamster and rabbit; a 0.5-nm hypsochromic shift in the guinea pig and no shift in the DBA/2N mouse; and (2) an increase in cytochrome P-450 apoproteins of the following molecular weights on sodium dodecyl sulfate-polyaerylamide gel electrophoresis: 54,000 and 55,000 in the C57BL/6N mouse; 48,000, 54,000 and 55,000 in the rat; 49,000 and 54,000 in the hamster; and 54,000 and 57,000 in the rabbit; a small increase in the 54,000 band was seen in the DBA/2N mouse and no increase in the guinea pig. In vitro addition of α-naphthoflavone selectively inhibited all four mono-oxygenase activities from the methylcholanthrene-treated C57BL/6N mouse, rat and hamster; 2-acetylaminofluorene N -hydroxylase and biphenyl 4-hydroxylase activities in the rabbit; and aryl hydrocarbon hydroxylase, 2-acetylaminofluorene N -hydroxylase and biphenyl 4-hydroxylase activities in the guinea pig. The addition of α-naphthoflavone enhanced the activities of aryl hydrocarbon hydroxylase and biphenyl 2-hydroxylase in liver microsomes from both control and methylcholanthrenetreated rabbits, but only biphenyl 2-hydroxylase activity was increased in the guinea pig: the activitity of 2-acetylaminofluorene N -hydroxylase was increased in both control and methylcholan-threne-treated DBA/2M mice, but only in the control C57BL/6N mouse. These data indicate that hepatic cytochrome P-448 is composed of multiple cytochromes, which differ among animal species, each catalyzing different mono-oxygenase activities.

Birth defects and aplastic anemia: differences in polycyclic hydrocarbon toxicity associated with the Ah locus.

The balance between cytochrome(s) P1-450 and other forms of P-450 in the liver, and probably many nonhepatic tissues as well, appears to be important in the toxicity, carcinogenicity, mutagenicity, and teratogenicity of numerous compounds. Thus, allelic differences in a single gene — the Ah locus —can have profound effects on the susceptibility of mice to drug toxicity and cancer. There is evidence for the Ah locus in the human.Striking increases in the incidence of stillborns, resorptions, and malformations caused by 3-methylcholanthrene or 7,12-dimethylbenz[a]anthracene were observed in the aromatic hydrocarbon “responsive” C57BL/6N, C3H/HeN, and BALB/cAnN inbred strains, compared with the genetically “nonresponsive” AKR/N. These data suggest that an association exists between the Ah locus and teratogenesis. Although numerous teratogenic differences among inbred mouse strains have been previously reported, this study is unique in that the genetic differences in teratogenicity observed were predicted in advance, on the basis of known differences in polycyclic hydrocarbon metabolism regulated by the Ah locus.Aplastic anemia induced by oral benzo[a]pyrene daily occurs in less than 4 weeks in the nonresponsive Ahd/Ahd individual, whereas his responsive Ahb/Ahb and Ahb/Ahd siblings remain healthy for 6 months while continuously receiving the same daily dose of benzo[a]pyrene. This phenomenon is probably associated with the “first-pass elimination” effect and the relatively high degree of aryl hydrocarbon hydroxylase induction in the bowel, liver, and bone marrow of the Ahb/Ahb or Ahb/Ahd mouse. A latency period of 2–4 weeks is demonstrated between the exposure of Ahd/Ahd mice to oral benzo[a]pyrene and death; morphological changes of toxicity are apparent early during this latency period. We propose that this animal model system might be useful in investigating human genetic differences in susceptibility to drug-induced aplastic anemia.

Effects of environmental chemicals on the genetic regulation of microsomal enzyme systems

The cytochrome P‐450‐‐mediated monooxygenase system, embedded in the endoplasmic reticulum of cell membranes, which metabolizes many hydrophobic environmental chemicals is involved in the metabolic potentiation and/or detoxification of many drugs and environmental pollutants. Examples of the interaction of such environmental chemicals with the genetic regulatory system controlling monooxygenase activity are presented, as well as the manner in which this interaction may be affected by genetic differences in susceptibility to drug toxicity and lead to chemically induced teratogenesis, mutagenesis, and carcinogenesis. Among the chemicals under study in this context were polycyclic hydrocarbons, halogenated hydrocarbons (Arochlor 1254, lindane, kepone), 2‐acetylaminojiuorene, and acetaminophen in inbred strains of mice and in genetic crosses. Only a very small number of genes was found to have a profound injiuence on an individuals increased susceptibility to cancer, mutation, and toxicity produced by different environmental chemicals. MUltiphasic response curves are predicted to occur with a given dosage of chemical causing different levels of toxicity in different individuals, depending upon the genetic predisposition of each individual. For example, cancer, chemical mutagenesis, hepatic necrosis, survival time, aplastic anemia, and possibly birth defects can vary among siblings in the same family because a relatively small number of genes regulates the differences in drug metabolism.