Regulation of CYP1A1 expression.

CYP1A1 is considered to be involved mainly in oxidative metabolism of exogenous chemicals and drugs. Synthesis of this hemoprotein is induced in livers, lungs, and other tissues of experimental animals by the administration of these chemicals. Regulatory mechanisms of the induction process of the protein have been investigated by the DNA transfer method using the isolated genomic DNA. At least two kinds of cis-acting regulatory DNA sequences are localized 5' upstream of the gene. One is distributed five times in a relatively wide range from -0.5 to -3.5 kb and functions as an inducible enhancer-designated xenobiotic responsive element or XRE. The other is localized just upstream of the TATA sequence and acts as a regulatory element for the constitutive expression. The two DNA elements are required for a high level of the inducible expression. Their cognate DNA binding factors are recognized in the nuclear extracts of Hepa-1 cells and rat liver cells which show the inducible expression of CYP1A1 in response to the inducer. This paper discusses the regulatory mechanisms of CYP1A1 gene expression by summarizing the present state of knowledge about properties of the DNA regulatory elements and their cognate DNA-binding factors.     

Regulation of xenobiotic and bile acid metabolism by the nuclear pregnane X receptor

The nuclear pregnane X receptor (PXR; NR1I2) is an integral component of the body's defense mechanism against chemical insult (chemoprotection). PXR is activated by a diverse array of lipophilic chemicals, including xenobiotics and endogenous substances, and regulates the expression of cytochromes P450, conjugating enzymes, and transporters involved in the metabolism and elimination of these potentially harmful chemicals from the body. Among the chemicals that bind and activate PXR is the toxic bile acid lithocholic acid; activation of PXR, in turn, protects against the severe liver damage caused by this bile acid.Thus, PXR serves as a physiological sensor of lithocholic acid and perhaps other bile acids and coordinately regulates genes involved in their detoxification. Interestingly, both the antibiotic rifampicin and the herbal antidepressant St. John's wort activate PXR and have anticholestatic properties, which suggests that more potent, selective PXR agonists may be useful in the treatment of biliary cholestasis or other diseases characterized by the accumulation of bile acids or other toxins in the liver.     

The unique role of rodents in the detection of possible human carcinogens and mutagens

Some of the probable reasons underlying the observation that not all chemicals shown to be genotoxic in vitro are capable of eliciting tumours in rodents or humans are discussed using appropriate examples. It is suggested that a substantial proportion of the resources currently available for conducting rodent carcinogenicity bioassays should be employed in the short-term evaluation in vivo of some of the many hundreds of chemicals recently defined as genotoxic in vitro, rather than in the protracted evaluation of a few chemicals, often of unknown activity in vitro, for carcinogenicity. A decision tree approach to the evaluation of chemicals for human mutagenic/carcinogenic potential is presented which is at variance with the construction and philosophy of many of the current legislative guidelines. The immediate need for the adoption of one of the available short-term in vivo liver assays, and/or the development of a short-term in vivo rodent assay capable of concomitantly monitoring different genetic end-points in a range of organs or tissues is emphasized.     

Purification and isoelectric heterogeneity of chicken tyrosinase

Alterations in rat liver transfer RNA (tRNA) methyltransferase activities have been observed after liver damage by various chemicals or by partial hepatectomy. The qualitative and quantitative nature of these activity changes and the time course for their induction have been studied. Since homologous tRNAs are essentially fully modified in vivo, E. coli tRNAs were used as in vitro substrates for the rat liver enzymes in these studies. Each of the liver-damaging agents tested rapidly caused increases in activities of the enzyme(s) catalyzing methyl group transfer to tRNAs that have an unmodified guanine at position 26 from the 5' end of the molecule. This group of tRNAs includes E. coli tRNANfmet, tRNAAla1, tRNALeu1, or Leu2, and tRNASer3 (Group 1). In each case N2-methylguanine and N2,N2-dimethylguanine represented 90% or more of the products of these in vitro methylations. The product and substrate specificity observed are characteristic of N2-guanine methyltransferase II (S-adenosyl-L-methionine : tRNA (guanine-2)-methyltransferase, EC 2.1.1.32). In crude and partially purified preparations derived from livers of both control and treated animals this enzyme activity was not diminished significantly by exposure to 50 degrees C for min. The same liver-damaging agents induced little or no change in the activities of enzymes that catalyze methyl group transfer to various other E. coli tRNAs that do not have guanine at position 26 (Group 2). The results of mixing experiments appear to rule out the likelihood that the observed enzyme activity changes are due to stimulatory or inhibitory materials present in the enzyme preparations from control or treated animals. Thus, our experiments indicate that liver damage by each of several different methods, including surgery or administration of chemicals that are strong carcinogens, hepatotoxins, or cancer-promoting substances, all produce changes in liver tRNA methyltransferase activity that represent a selective increase in activity of N2-guanine tRNA methyltransferase II. It is proposed that the specificity of this change is not fortuitous, but is the manifestation of an as yet unidentified regulatory process.     

Use of yeast respiratory adaptation test system to detect chemical mutagens/carcinogens in mammals.

An approach to follow distribution of injected DNA-acting chemicals (mutagens/carcinogens) in animal tissues has been described. This is based on the use of respiratory adaptation (mitochondrial biogenesis) process in Saccharomyces cerevisiae during transition from anaerobic to aerobic state. By virtue of specific interaction of such chemicals with mitochondrial DNA associated with promitochondrial structures this process is extremely sensitive to DNA-acting chemicals. Solutions of berylium sulphate, aflatoxin G1, aflatoxin B1, and carbaryl (all known DNA-acting agents) were injected to rats at low concentrations and, after 24 hr, distribution of these chemicals or their metabolites was studied by determining the inhibitory action of appropriately diluted urine and tissue homogenates on respiratory adaptation in S.cerevisiae. Detectable amounts of the chemicals and their DNA-acting metabolites could be analyzed in urine, liver, lungs, kidney and spleen.     

In Vivo measurement of unscheduled DNA synthesis and S-phase synthesis as an indicator of hepatocarcinogenesis in rodents

Measurement of chemically induced DNA repair as unscheduled DNA synthesis in rodent liver following in vivo treatment is a useful screen for potential hepatocarcinogens. In addition to measurement of unscheduled DNA synthesis, examination of S-phase synthesis provides an indicator of chemically induced cell proliferation in the liver, which may be a basis for hepatic tumor promotion. Several chemicals and classes of chemicals have been examined using these endpoints. The pyrrolizidine alkaloid riddelline is a potent genotoxic agent in vitro, and in vivo studies confirm this response as riddelline induces significant elevations in unscheduled DNA synthesis and S-phase synthesis in rat liver. Conversely, H. C. Blue dyes #1 and #2 are both potent genotoxic agents in vitro but fail to express this genotoxicity in vivo. H. C Blue #1 induces significant increases in S-phase synthesis in B6C3F1 mouse liver, which correlates with the observed carcinogenicity of this compound. Halogenated hydrocarbons likewise fail to induce unscheduled DNA synthesis in vivo, but many of these compounds do increase hepatic cell proliferation in mice, which may be the principal mechanism of hepatocarcinogenesis in this species.Abbreviations BCMEE bis(2-chloro-l-methylethyl)ether - dThd thymidine - HCB1 H.C. Blue #1 - HCB2 H.C. Blue #2 - UDS unscheduled DNA synthesis     

Methods for testing chemical additives to'prevent moulding of hay

Three methods for testing additives for their ability to prevent moulding of hay are described. Storage of 25 g samples of hay in glass jars at 25 ^oC was useful for screening many chemicals rapidly. Dewar flasks (4–5 1) allowed testing on 500 g hay samples with sufficient insulation to allow any effects of chemicals on spontaneous heating as well as on moulding to be measured. Large drums containing 20 kg hay were used for larger scale tests with partially dried fresh, or rewetted old hay. Proprietary additives were unsuccessful at their recommended application rates and some even failed to show antifungal activity with very much larger doses. Volatile fatty acids, particularly propionic acid, and ammonium propionate were the most effective chemicals tested. The amount of propionic acid needed to prevent moulding and spontaneous heating could be defined in terms of the amount of water in the hay. Where the ratio of propionic acid to water was greater than 1–25:100 heating was rare. Ammonium propionate was slightly less effective than propionic acid against moulding. However, it lacks a pungent odour, is less volatile, less corrosive and is more pleasant and safer to handle.     

Tissue-specific expression and subcellular distribution of murine glutathione S-transferase class kappa.

Class kappa glutathione S-transferases are a poorly characterized family of detoxication enzymes whose localization has not been defined. In this study we investigated the tissue, cellular, and subcellular distribution of mouse glutathione S-transferase class kappa 1 (mGSTK1) protein using a variety of immunolocalization techniques. Western blotting analysis of mouse tissue homogenates demonstrated that mGSTK1 is expressed at relatively high levels in liver and stomach. Moderate expression was observed in kidney, heart, large intestine, testis, and lung, whereas sparse or essentially no mGSTK1 protein was detected in small intestine, brain, spleen, and skeletal muscle. Immunohistochemical (IHC) analysis for mGSTK1 revealed granular staining of hepatocytes throughout the liver, consistent with organelle staining. IHC analysis of murine kidney localized GSTK1 to the straight portion of the proximal convoluted tubule (pars recta). Staining was consistent with regions rich in mitochondria. Electron microscopy, using indirect immunocolloidal gold staining, clearly showed that mGSTK1 was localized in mitochondria in both mouse liver and kidney. These results are consistent with a role for mGST K1-1 in detoxification, and the confirmation of the intramitochondrial localization of this enzyme implies a unique role for GST class kappa as an antioxidant enzyme.     

Helical polysomes induced by aflatoxin B1 in vivo : A new hypothesis for helix formation by chemicals and carcinogens

We have studied the induction of helical polysomes by aflatoxin B₁ in liver and kidney cells from rat and mouse. We succeeded in giving to reticulocyte polysomes a shape resembling helices after in vitro treatment with O-methylthreonine which is used as an inhibitor of polypeptide chain termination. From this and knowing the site of action of aflatoxin B₁ on rat liver polysomes, we hypothesize that the induction of helical polysomes in tissues from adult animals treated by chemicals or carcinogens is due to the inhibition of release of ribosomes from the messenger RNA (mRNA). Theoretical studies of protein synthesis inhibition are in agreement with this new hypothesis.     

Post-translational modifications of mitochondrial aldehyde dehydrogenase and biomedical implications

Aldehyde dehydrogenases (ALDHs) represent large family members of NAD(P)+-dependent dehydrogenases responsible for the irreversible metabolism of many endogenous and exogenous aldehydes to the corresponding acids. Among 19 ALDH isozymes, mitochondrial ALDH2 is a low Km enzyme responsible for the metabolism of acetaldehyde and lipid peroxides such as malondialdehyde and 4-hydroxynonenal, both of which are highly reactive and toxic. Consequently, inhibition of ALDH2 would lead to elevated levels of acetaldehyde and other reactive lipid peroxides following ethanol intake and/or exposure to toxic chemicals. In addition, many East Asian people with a dominant negative mutation in ALDH2 gene possess a decreased ALDH2 activity with increased risks for various types of cancer, myocardial infarct, alcoholic liver disease, and other pathological conditions. The aim of this review is to briefly describe the multiple post-translational modifications of mitochondrial ALDH2, as an example, after exposure to toxic chemicals or under different disease states and their pathophysiological roles in promoting alcohol/drug-mediated tissue damage. We also briefly mention exciting preclinical translational research opportunities to identify small molecule activators of ALDH2 and its isozymes as potentially therapeutic/preventive agents against various disease states where the expression or activity of ALDH enzymes is altered or inactivated.     

A simple, sterile food source for rearing the larvae of Lucilia sericata (Diptera: Calliphoridae)

arvae of the sheep blowfly Lucilia sericata (Meigen) were reared on various mixtures of puréed liver, agar, brewer's yeast and trypsin, in order to develop a simple, sterile, tissue-based diet. Growth and survival rates of larvae reared on a sterile 1:1 mixture of puréed liver with 3% Bacto agar equalled or exceeded those of larvae reared on raw liver. The addition of yeast and/or trypsin to the medium was of no additional benefit. This sterile, homogenous, tissue-based substrate offers a simple, convenient, inexpensive growth medium for rearing larvae for maggot therapy, and for testing the effects of various chemicals and dietary constituents on necrophagous insect larvae. It may be useful, therefore, in studies of myiasis, forensic entomology, and toxicology. This rearing medium also has the advantage that it can be stored for many months at room temperature without progressive decomposition or offensive odour.     

Long-term programming of skeletal muscle and liver lipid and energy metabolism by resveratrol supplementation to suckling mice

Metabolic programming by dietary chemicals consumed in early life stages is receiving increasing attention. We here studied long-term effects of mild resveratrol (RSV) supplementation during lactation on muscular and hepatic lipid metabolism in adulthood. Newborn male mice received RSV or vehicle from day 2–20 of age, were weaned onto a chow diet on day 21, and were assigned to either a high-fat diet (HFD) or a normal-fat diet on day 90 of age for 10 weeks. RSV-treated mice showed in adulthood protection against HFD-induced triacylglycerol accumulation in skeletal muscle, enhanced muscular capacities for fat oxidation and mitochondria activity, signs of enhanced sirtuin 1 and AMP-dependent protein kinase signaling in muscle, and increased fat oxidation capacities and a decreased capacity for lipogenesis in liver compared with controls. Thus, RSV supplementation in early postnatal life may help preventing later diet-related disorders linked to ectopic lipid accumulation in muscle and liver tissues.     

Role of receptors in human and rodent hepatocarcinogenesis

The liver is a major target organ in rodent carcinogenicity assays. Amongst the agents that are effective in producing rodent liver tumours are many chemicals which are not mutagenic, but are believed to mediate their effects by promoting the clonal outgrowth of initiated cells. Some of these chemicals, such as dibenzo-p-dioxins and certain PCBs, have been demonstrated to interact with specific cellular receptors and receptor binding appears crucial for their tumourigenic activity. Enzyme-altered foci in rat liver may serve as a sensitive means to estimate the promoting activity of these agents in rodents. Mechanistic considerations are of relevance when extrapolating these date from rodents to humans.     

Pest control in museums: toxicity of para-dichlorobenzene, ‘Vapona’™, and naphthalene against all stages in the life-cycle of museum pests,Dermestes maculatus Degeer,and Anthrenus verbasci (L.) (Coleoptera: Dermestidae)

Museums, herbaria, libraries and archival collections have traditionally relied on chemicals for the prevention and treatment of pest infestations. While current evidence suggests that the use of chemicals is declining, however, they are still found in many collections. The efficacy of three ‘insecticides’, para-dichlorobenzene, ‘Vapona’ and naphthalene (used in some museums to treat localized infestations and for their apparent residual benefit) against two insect species (Coleoptera: Dermestidae) was evaluated. Despite considerable variation in insect susceptibility, ‘Vapona’ was found in general, to be the most effective of the three chemicals used, particularly against larvae and adults. Naphthalene was the least effective, with low mortality rates recorded in the majority of the insect stages tested. Based on this study, an exposure/mortality relationship is presented for the prevention and treatment of insect infestations in museum and archival collections.     

Abnormal liver function associated with occupational exposure to dimethylformamide and glutathione S-transferase polymorphisms

Dimethylformaide (DMF) is a major solvent predominately used in synthetic leather and resin production. Many human and animal studies have linked the cause of hepatoxicity to DMF. Previously, the authors demonstrated the significant dose–response relationship between abnormal liver function tests and DMF exposure and the interaction with hepatitis B virus (HBV) infection in Taiwanese workers. Because the toxic effect of various chemicals can be modified by metabolic traits, the study also investigated the influence of the glutathione S-transferases (GSTM1 and GSTT1) on the toxic effect of DMF. The average DMF exposure concentration was 23.87 ppm (range 5.2–86.6 ppm) in the high-exposure (≥5 ppm) group and 2.41 ppm (range 0.9–4.3 ppm) in the low-exposure (<5 ppm) group. There were 13 of 44 (29.6%) abnormal liver function tests (elevations of either glutamate oxaloacetate transaminase (GOT) or glutamate pyruvate transaminase (GPT)) among the high DMF exposure workers, two of 22 (9.1%) abnormal liver function tests among the low DMF exposure workers. Chronic liver disease as determined by ultrasonography was present in seven of 44 (15.9%) high DMF exposure workers, and 0 of 22 (0%) low DMF exposure workers. There were 11 of 34 (32.4%) abnormal liver function tests among the GSTT1 null genotype workers, and four of 32 (12.5%) abnormal liver function tests among the GSTT1-positive genotype workers. Compared with the low DMF exposure workers, the adjusted odds ratio and 95% confidence intervals for abnormal liver function tests was 6.78 (0.94–48.7) for the high DMF exposure workers. Compared with the GSTT1-positive genotype workers, the adjusted odds ratio and 95% confidence intervals for abnormal liver function tests was 4.41 (1.15–16.9) for the GSTT1 null genotype workers. Compared with the low DMF group with GSTT1-positive genotype workers, the odds ratio (adjusted for HBV status) of abnormal liver function test was 12.38, 95% CI=(1.04–146.9) for the high DMF group with GSTT1 null genotype workers. This study indicates that abnormal liver function and chronic liver disease are associated with DMF exposure, and there are more than multiplicative interaction effects on abnormal liver function tests between the DMF exposure and the GSTT1 genotype.     

Effects of safrole and isosafrole pretreatment on N- and ring-hydroxylation of 2-acetamidofluorene by the rat and hamster

1. The effects of safrole and isosafrole pretreatment on both N- and ring-hydroxylation of 2-acetamidofluorene were studied in male rats and hamsters. 2. Isosafrole (100mg/day per kg body wt.) pretreatment of rats for 3 days did not have any effect on urinary excretion of hydroxy metabolites of 2-acetamidofluorene. However, similar pretreatment with safrole produced increased urinary excretion of N-, 3- and 5-hydroxy derivatives. 3. Similar treatment with these two chemicals for 3 days increased ring-hydroxylation activity by rat liver microsomal material. Increases in N-hydroxylation were much less than those in ring-hydroxylation. Isosafrole was twice as effective as safrole. 4. Increases in hydroxylating activity due to safrole or isosafrole treatment were inhibited by simultaneous administration of ethionine. Similarly, ethionine inhibition was almost completely reversed by the simultaneous administration of methionine. 5. Safrole or isosafrole (0.1mm and 1mm) inhibited 7-hydroxylation activity by liver microsomal material from control rats. At 1mm these two chemicals inhibited both 5- and 7-hydroxylation activity by liver microsomal material from 3-methylcholanthrene-pretreated rats. 3-Hydroxylation activity was not inhibited by 1mm concentrations of these two chemicals. 6. A single injection of safrole (50100 or 200mg/kg body wt.) 24h before assay had no appreciable effect on either N- or ring-hydroxylation activity by hamster liver microsomal material. However, isosafrole (200mg/kg body wt.) treatment inhibited N-, 3- and 5-hydroxylation activities by hamster liver microsomal material; it had no effect on 7-hydroxylation activity.