Diane J. Benford

MECHANISMS OF TOXIC INJURY

2. The rate of production of initiating factor(s) and its rate of detoxication. 3. The accessibility of the initiating factor to the crucial “target” cellular molecules and the extent and persistence of the reaction between them. 4. The biological role of these “target” molecules (which may vary with cell type, physiological state of the cell, etc.). 5. The ability of cellular defense mechanisms to repair, replace, or compensate for these altered “target” molecules. 6. The nature and extent of release of products from the injured cell that may cause cytotoxic or stimulatory effects on other cells of the same or different cell types. initiating factor.

Investigations of the genotoxicity and cell proliferative activity of dichlorvos in mouse forestomach

This study investigated the possible mechanism by which dichlorvos may have caused forestomach tumours in mice in a chronic corn oil gavage cancer bioassay [NTP (1989) Toxicology and carcinogenesis studies of dichlorvos in F344/N rats and B6C3F1 mice (gavage studies). National Toxicology Program Technical Report 342, NIH Publ. No 89-2598]. For this purpose, a method has been developed to assess the genotoxicity of irritant substances on mouse forestomach epithelium. Groups of five B6C3F1 mice were given a single oral dose of dichlorvos, the genotoxic forestomach carcinogen 1-methyl-3-nitro-1-nitrosoguanidine (MNNG) or the irritant, non-genotoxic forestomach carcinogen butylated hydroxyanisole (BHA). After periods of 2-48 h, three parameters were assessed: unscheduled DNA synthesis (UDS) by autoradiography of tissue sections, replicative DNA synthesis (RDS) also by autoradiography of incorporated [3H]thymidine, and histopathological changes, including hyperplasia. MNNG induced UDS but not RDS or hyperplasia in forestomach epithelium, consistent with its genotoxic mode of action. BHA and dichlorvos did not induce UDS, consistent with absence of genotoxic activity in the forestomach after in vivo exposure. In contrast, BHA and dichlorvos induced RDS and subsequent hyperplasia, which is likely to result from irritant damage. These data suggest that the chronic effects of dichlorvos on mouse forestomach epithelium in the oral gavage bioassay were mediated via enforced cell proliferation, rather than by a genotoxic mechanism.

Activation of hepatic microsomal biphenyl 2-hydroxylation by corticosteroids.

1. 4-Hydroxylation was a major route of biphenyl metabolism in liver microsomes from control and phenobarbitone-pretreated rats, with 2- and 3-hydroxybiphenyl as lesser metabolites. 2. Many corticosteroids, when added to the microsomal incubation mixture, selectively increased 2-hydroxylation with little or no effect on 3- and 4-hydroxylation. Betamethasone caused the greatest activation (400%). 3. In liver microsomes from controls hamsters and 3-methylcholanthrene-pretreated rats, the basal hydroxylase activity, especially 2-hydroxylation, was much higher, but the quantitative increase following betamethasone addition was similar to that in liver microsomes from control and phenobarbitone-pretreated rats. 4. Pretreatment of rats with betamethasone also resulted in a small increase in biphenyl 2-hydroxylation activity after 4 h, returning to control values after 6 h. 5. In vitro addition of estradiol or testosterone had no effect on either basal or betamethasone-activated biphenyl 2-hydroxylation.

Characterization of the activation of hepatic microsomal hydroxylation by betamethasone and α naphthoflavone

Abstract Biphenyl 2-hydroxylation is selectively activated in vitro by incubation of betamethasone or α naphthoflavone with control male rat liver microsomes. Biphenyl 3- and 4-hydroxylation activities are unchanged or marginally inhibited. The nature of the enzymes involved in the activation has been investigated. Metyrapone (1 mM) completely inhibited the expression of the activation but had a lesser effect on the basal 2-, 3- and 4-hydroxylation activities. SKF525A (1 mM) † inhibited both basal and betamethasone-activated enzyme activities by 25–35 per cent. Of other drug metabolizing enzymes investigated, only benzo[ a ]pyrene hydroxylation activity was increased by betamethasone and α naphthoflavone. Acetone (0.6M) caused a small activation (40 per cent) of biphenyl 2-hydroxylation but inhibited 4-hydroxylation. The non-ionic detergent Brij 35 inhibited biphenyl 2-, 3- and 4-hydroxylation. It was concluded that activation of biphenyl 2-hydroxylation differs from activation of aromatic amine hydroxylation and glucuronyl transferase but may be related to activation of benzo[ a ]pyrene hydroxylation by naphthoflavones.

Metabolizing systems in cell culture cytotoxicity tests

1. The addition of 9000 g supernatant of rat liver homogenate (S9) or rat liver microsomal fractions to a cytotoxicity test system using BCL-D1 cells has been investigated. 2. The choice of culture medium influenced the intrinsic cytotoxicity of the metabolising system to the BCL-D1 cells. Use of Hams F10 nutrient mixture resulted in greater cytotoxicity compared with several other media. 3. Microsomal fractions provided greater cytochrome P-450 dependent activation of cyclophosphamide and were less cytotoxic than S9. 4. Direct-acting toxic compounds, such as p-aminophenol, were less toxic in the presence of a metabolising system. This was due to protein-binding rather than enzymic detoxification.

The design and use of in vitro toxicity tests

It is timely to consider critically our present approach to toxicity testing. On the one hand there is considerable public concern over the number of chemicals for which inadequate toxicological information exists and to which man is significantly exposed. Estimates that 30,000–50,000 chemicals are in this category have been made (e.g. Miller, 1978). In addition, several hundred new chemicals are being introduced each year which require some form of toxicological investigation. On the other hand there are mounting economic and moral pressures to change the testing methods that are currently employed.

Mechanistic studies on the activation of biphenyl 2-hydroxylation by glucocorticoids

In order to establish the mechanism by which the selective activation of biphenyl 2-hydroxylation by betamethasone occurs the effect of modifying possible critical factors in the hydroxylation process has been examined. Activation of biphenyl 2-hydroxylation by betamethasone was found in detergent-solubilized rat liver microsomes indicating that intact microsomal membranes are probably not necessary for the activation. Betamethasone had no effect on the spectrally apparent binding of biphenyl or of other type I, type II or reverse type I model substrates. The activation process did not appear to be greatly influenced by changing the ratio of cytochrome P-450 reductase to cytochrome P-450 nor by changing the amount of NADPH. Addition of NADH increased the extent of activation suggesting that betamethasone facilitates transference of the second electron to cytochrome P-450. However, betamethasone also stimulated cumene hydroperoxide supported biphenyl 2-hydroxylation; therefore a step subsequent to cytochrome P-450 reduction is also involved in the activation. Activation did not correlate with increased uncoupling of an active oxygen-cytochrome P-450 complex to form hydrogen peroxide.

Receptor-Mediated Transfer of Proteins from Blood to Bile

Bile proteins derive from several different sources. Firstly there is a small, mol. wt.-dependent, leakage of proteins across the tight junctions between liver cells. Secondly, proteins are solubilized from the bile-canalicular face of hepatocytes and from the luminal face of bile-duct lining cells. Thirdly there is discharge of the contents of lysosomes into bile. Finally there is selective transport of proteins across hepatocytes from blood to bile, the mechanism of which we have established by methods described in this article.

THE RAPID ACTIVATION OF P-450-MEDIATED METABOLISM BY CORTICOSTEROIDS

Rapid activation of hepatic microsomal biphenyl 2-hydroxylation may be produced by a range of synthetic and naturally occurring corticosteroids of which betamethasone is the most effective. The activation, which is relatively selective for certain aromatic hydroxylation reactions, probably involves a particular form of P-450 which is normally latent in hepatic microsomes whether obtained from control, phenobarbitone or 20-methylcholanthrene pretreated rats.