The role of MT in Cd toxicology has become clearer by the use of MT-I transgenic and MT-I and -II knock-out animals. We have shown that:(1)MT-transgenic and -null mice have altered tissue MT protein levels;(2)MT-transgenic and-null mice appear to be normal in other detoxifyng systems examined, except for slight alterations in tissue Zn concentration;(3)MT does not appear to inhibit Cd absorption from the gestrointestinal tract, nor affect Cd tissue distribution;(4)MT reduces the elimination of Cd from liver;(5)MT protects against acute inorganlc Cd-induced lethality and hepatotoxicity, and the mechanism of the protection appears to be due to its ability to sequester Cd in the cytosol, thus reducing the amunt of Cd in critical organelles;(6)MT modulates Cd-induced expression of protooncogene(c-jin) and tumor suppress genes(p53) in mouse liver;(7)MT does not protect against CdMT-induced acute renal injury, and Zn-induced protection against CdMT-induced acute nephrotoxicity does not appcear to be mediated through MT;(8)Chronic Cd administration produces renal injury inb MT-null mice, indicating that Cd-induced nephrotoxicity is not necessarily mediated through the CdMT complex;(9)MT protects against chronic CdC12 nephropathy, suggesting that intracellular MT is an important adaptive mechanism decreasing CdC12 nephrotoxicity, and that a single injection of CdMT may not be a good model to study chronic Cd nephropathy;(10)genetic background of mouse strains, rather than constitutive MT levels, is a more important determinant for Cd-induced acute testicular injury. In addition to Cd detoxication, MT-transgenic and MT-null mice are also good models to determine other functions of MT. MT plays important roles in maintaining Zn homeostasis and protection against Zn toxicity. Knock-out of the MT gene also renders animals/cells more vulnerable to oxidative stress and DNA alkylating agent-induced toxicity. Therefore, the MT-transgenic and knock-out mouse models provide complementary approaches to those used previously, and have greatly increased our understanding of the role of MT in Cd toxicology, as well as other biological functions of MT.

View full abstract

A medium-term liver bioassay system for rapid detection of carcinogenic agents using male F344 rats has been developed, in order to bridge the gap between long-term carcinogenity tests and short-term screening assays. The system is fundamentally based on the two-stage hypothesis of oarcinogenesis : intiation with diethylnitrosamine (200 mg/kg bw, ip) is follwed by test chemical administration during the second, in combination with 2/3 partial hepatectomy. It requires only 8 weeks for animal experimental treatment and a further few weeks for quantitative analysis of immunohistochemically-demonstrated glutathione S-transferase placental from positive hepatic foci. A total of 291 chemicals/substances have already been analyzed in this laboratory and the efficacy of the system for hepatocarcinogens has thereby been well established. This bioassay is particularly useful for dose-response and chemical mixture studies, usually requiring large0scale experiments and also for evaluation of chemoperventive agents. Another bioassay, a medium-term multiorgan bioassay system, using 5 different chemical carcinogens, diethylnitrosamine (DEN), N-methylnitrosourea (MNU), N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN), 1, 2-dimethylhydrazine (DMH) and 2, 2-dihydroxy-di-n-propylnitrosamine (DHPN), has also been established for rapid detection of not only hepaticatcinogens, but also other organ-target carcinogens. Rats were initially treated with a single ip administration of 100 mg/kg DEN, 4ip administrations of 20 mg/kg MNU, 4 sc doses of 40 mg/kg DMH for 2 weeks and then 0.1% DHPN for 2 weeks. Test chemicals are administered after the carcinogens exposure. Animals were sacrificed at the end of week 36, and major organs were examined histologically. Carcinogenic activities of test chemicals were compared between the test chemical treated group and carcinogen exposures group (control froup). It is increasingly becoming regarded that these bioassays are useful methods and are appropriate alternative tests systems for carcinogencity risk assessment. Therefore, 'the International Conference on Harmonization (ICH) of Technical Requirements for the Registration of Pharmaceuticals for Human Use' has proposed that these two bioassays can be used as "additional tests for cercinogenic activity in vivo."

View full abstract

Following expression of UDPGTh1 and UDPGTh2 in Cos-1 cells, each isoform metabolized thiree types of dihyroxy-or trihydroxy-substituted ring structures, including the 3, 4-catechol estrogen (4-hydroxyestrone), estriol and 17-epiestriol, and hyodeoxycholic acid(HDCA), but the UDPGTh2 isozyme was 100-fold more efficent than UDPGTh1. UDPGTh1 and UDPGTh2 are 86% identical overall (76 differences out of 528 amino acids), including 55 differences in the first 300 amino acids of the amino terimnus, a domain which confers isoform substrate specificity. The data indicate a high level of conservation in the amino terminus is not required for the presevation of substrate specificity. Analysis of glucuronidation activity encoded by UDPGTh1/UDPGTh2 chimeric cDNAs constructed at their common restriction sites, Sac I (codon 279), Nco I (codon 385), and Hha I (codon 469), showed that nine amino acids between residues 385 and 469 are important for catalytic efficiency, suggesting that this region represents a domain which is critical for actalysis but distinct from that responsible for aglycon selection. Screening of leukocyte DNA cosmid library with human UDPGT-Br1 (1-470bps) or UDPGT-Br2 (1-450bps) resulted in three overlapping clones, which were isolated and mapped by endonucleases. Construction of subclones and DNA sequencing, Southern blot analysis revealed that a cluster of 4 exons (132, 88, 220, 1, 032, bps in one clone) encodes the entire region of 3' identity shared between human UDPGT-phenol, human UDPGT-Br1 and human UDPGT-Br2. A similar strategy but using probes which correspond to the unique regions of human UDPGT-Br1 and human UDPGT-Br2 showed that the exon 1 of UGTID encodes the unique region of human UDPGT-Br1, and human UDPGT-Br2 and is located 5.6 and 49 Kb, respectively, upstream of the 4 common exons.

View full abstract

Epidemiological studies have demonstrated a strong association between exposure to AFB1 and an increased incidence of human hepatocellular carcinoma (HCC). This association has led to a need for accurate techniques relating AF exposure to an individual's risk of developing disease. With the understanding of the progressive processes of carcinogenesis, opportunities for the identification of molecular biomarkers reflecting events from exposure thorugh clinical disease are provided. However, the development of biomarker methods to monitor human exposure to AFs requires techniques which are sensitive, specific, and amenable to large numbers of samples. To better understand the role of AF exposure with respect to HCC incidence, immunoassays for the biological quantitation of free AFB1, its metabolites, and its adduct macromolecules have been developed. ELISA appears to offer a suitable for use in epidemiological studies for monitoring short-term exposure to AFs, as it has the appropriate sensitivity and specififity. However, the presence of substances that are presumably not AFs and which are inhibitory in the ELISA system has necessitated the development of purification techniques, usually based on adsorption onto Sep-Pak C18 cartridges and immunoaffinity chromatography. Many protocols have been development for the assay of soluble AF metabolites in urine, milk and blood. However, these assays only indicate recent exposure, whereas the presence of albumin-AFB1 adducts in peripheral blood could present a useful material for assessing longer-term exposure. Among the various possible biomarkers of AF exposure, the measurements of AF-DNA and-protein adducts are of major interest because they are direct products of damage to a critical cellular macromolecular target. In Thailand, AF contamination of foods was reported to be high. More recent data using biomarkers as measures of AF exposure will be discussed. The data from epidemiological studies, AF exposure assessment using AF-albumin adduct and urinary AF level as exposure markers as well as the prevalence of p53 mutation at codon 249 are all suggestive of a limited importance of AF in the etiology of HCC in this country compared to other areas, including parts of Africa and China. These results also indicate that research on other potential hepatocarcinogens should not be neglected.

View full abstract

The ubiquitous mycotoxin ochratoxin A (OTA) is found as a frequent contaminant of a large variety of food and feed and beverage such as beer, coffee and win. It is produced as a secondary metabolite of moulds from Aspergillus and Pennicillium genera. Ochratoxin A has been shown experimentally to inhibit protein synthesis by competition with phenylalanine its structural analogue and also to enhance oxygen reativr radicals production. The combination of these basic mechanisms with the unusual long plasma half-life time (35 days in non-human primates and in humans), the metabolisation of OTA into still active derivatives and glutathion conjugate both potentially reactive with cellular macromolecules including DNA could explain the multiple toxic effects, cytotoxicity, teratogenicity, genotoxicity, mutagenicity and carcinogenicity. A relation was first recognised between exposure to OTA in the Balkan geographical area and Balkan Endemic Nephropathy (BEN) with a high incidence (nearly 50 times higher than normal) of urinary tract tumours. Exposure rates of OTA are measurable in blood of humans and animals and are established in several countries including Scandinavia, Germany, France, Italy, Canada, Japan and Northern Africa mainly Tunisia and Egypt. The impact of OTA exposure in non-endemic areas in the world is not known, the rates of exposure being not correlated with the disease records, especially in developed countries, due to lake of well-designed epidemiological studies, genetic polymorphism and maybe to dietary contents of radical scavengers and antioxidants. However the incidence and mortality rates of renal cancer are increasing in European countries and Northern Africa which could be a global resultant of human exposure to natural compounds food such as mycotoxins and especially ochratoxin A. In addition to special care to prevent the growth of moulds and detoxification measures there was a need for the prevention of the OTA-induced toxic effects once the toxin is ingested. For this purpose several compound have been studied including some therapeutic agents such as piroxicam which cannot be proposed for a large scale use in humans for preventive pupose. Among other compounds, Aspartame, already used as sweetener has shown a real effectiveness in vivo confirmed largely in vitro. When rats exposed to OTA (289 μg/kg) by oral route every two days are given 25 mg/kg similarly for several weeks, all the toxic effects including genotoxicity are very efficiently prevented as shown for example by the disappearance of DNA-adducts in tissues excised from treated animals. Aspartame is also effective in washing out the toxin when given afterwards to animals intoxicated by the same oTA doses for several weeks. In vitro, provided that it is added in cell culture medium before OTA it prevent significantly the inhibition of protein synthesis and lipid peroxidation induced by the toxin. Obviously the molecular mechanism mediating the preventive effect of Aspartame is the delivery of phenylalanine by cleavage of the peptide but also the direct effect of the peptide on the bending capacity and transport of the toxin in vivo and in yitro. As a matter of fact when Aspartame is given to animals or added in culture medium the amount of peptide found unchanged (10-15%) may account for a preventive effect as entire peptide.

View full abstract

The NMDA receptor non-competitive antagonist, [³H] MK-801, was used as a ligand for an autoradiographic study to determine the effects of lead on NMDA receptor in rat brain. Adult male rats were given lead acetate, 100 mg/kg, or sodium acetate, 36 mg/kg (control), by i. p. for 7 days. Lead levels were detected in blood (41.1μg/dl) and brain (16.7-29.4μg/g). Concentrations of lead in various brain regions did not differ. [³H] MK-801 binding was heterogeneous throughout the brain with the following order of binding densities : hippocampal formation>cortex>caudate-putamen>thalamus>brainstem. Lead exposure caused a decrease in [³H] MK-801 binding to NMDA receptors in the hippocampal formation including CA2 stratum radiatum, CA3 stratum radiatum and presubiculum, and in the agranular insular, cingulate, entorhinal, orbital, parietal and perihinal areas of cerebral cortex. In another experiment, female rates were exposed pre-and post-natally from the 4th±1 post cconception day with 1, 000 ppm lead in their drinking water. This treatment continued after weaning. No effects of lead on [³H] MK-801 binding were found at postnatal day (PM) 28. However, lead caused a significant increase in [³H] MK-801 binding in the hippocampus including CA1 and CA2, and in the occipital and temporal cortical areas at PN 56 and at PN 112. Increases in [³H] MK-801 binding were also found in entorhinal cortex and dentate gyrus at PN 112. The hippocampal formation is a critical neural structure for learning and memory processes, whereas cortical and subcortical regions are involved in the modulation of complex behavioral processes. NMDA receptors have been shown to play a key role in synaptic plasticity underlying learning and memory. Therefore, lead-induced alterations of ligand binding to NMDA receptors in the hippocampal formation and cortical areas may play a role in lead-induced neurotoxicity.

View full abstract

Lead has been known to be toxic to most living things at high dose. It is found naturally in earth and present in almost all parts of the environment, such as foods, air, water, dust, soil, paint, and tissues of living organisms including human. This metal is being used in various aspects including the manufacturing of storage batteries, production of chemicals, paints and gasoline additives. It is also used to make various metal products, e. g. sheet lead, solder, and pipes. Human exposure to lead is mainly from foods and other environments. However, it is expected that exposure to environmental lead is normally exessive and produces toxic effects. The well-known and exessive environmental exposures are air of industrial and heavy traffic areas. Use of leaded gasoline has caused the main lead pollution for years in almost every big city. Therefore, city inhabitants normally exposed to lead much more than those who live in the rural area. The most vulnerable groups at risk to lead exposure are fetuses and preschool age children. Young children in the 2-3 year-old age may be the most at risk for exposure to contaminated soil. Adults are affected when exposure is exessive in the working place and causing lead poisoning. Toxicities are mainly on heme biosynthesis, neurological effects including encepharopathy, peripheral neuropathy, and most importantly on I. Q. deficits. It also affects renal tissues to produce acute and chronic nephropathy and elevated blood pressure. There are studies of lead exposure of various means and the effects on human health, both in children and adults. Lead in environment and human exposure are expected to stay with us for long to come, due to the still required lead use in many fields, particularly the use of lead in storage batteries and others. The magnitude of exposure will depend solely on the control of use by not allowing the contamination of lead in our environment to be excessive.

View full abstract

The rapid industrialization in Taiwan has caused both prosperity and environmental pollution. The purpose of this study is to demonstrate a case of both occupational and exvironmental lead poisoning. A patient of lead poisoning intiated a survey of the battery recycling factory, which revealed that 31 of 64 workers suffered from lead poisoning. Children who attended a nearby kindergarten showed a significant increase of blood lead up to 15-25 μg/dl and a mild but significant decrease of IQ (intelligent quotient, by Binet-Simon scale) if compared with children of a nonexposed but socioeconomically comparable kindergarten. Outdoor workers of the nearby forging factory also showed a significant increase of blood lead if compared with indoor workers or workers of another nonexposed forging factory 20 Km away. Air sampling showed an average of more than 10 μg/m³ in the kindergarten. Soil sampling and analysis also revealed 400 folds increase of lead content, which decreased if the sample was taken deep down to 15-30 cm or 350 meters away from the battery recycling smelter. Moreover, after children were moved away from the pollution source, follow-up examination performed 2.5 years later showed a significant decrease of blood lead and partial recovery of IQ among them.

View full abstract

More than 20, 000 passengers of Tokyo underground trains were intoxicated with warfare toxic chemicals. Most of the patients examined had marked miosis and decreased serum cholinesterase activity. Transient increase of serum CDK activity after 3 days of the exposure was the another sign. We intensively analyzed the metabolites in the urine of 4 patients. The following analytic results indicated the exposure to sarin as well as contaminated compounds such as diisopropyl methylphosphonate (DIMP), ethyl methylphosphonate fluoridate (EMPF, or ethylsarin), diethyl methylphosphonate (DEMP), and ethyl isopropyl methylphosphonate (EIMP). (1) Isopropanol (IPA) and ethanol (EtOH) were detected of large quantities in the urine samples, and were thought to be derived from sarin and the sarin counterpart, EMPF, DIMP, DEMP and EIMP. (2) Monoalkyl methylphosphonic acids (isopropyl methylphosphonic acid (IMPA) and ethyl methylphosphonic acid (EMPA) also were excreted in large amounts with taking the simlllllllllllllar excretion pattern of IPA and EtOH. (3) The metabolite only derived from sarin and ethylsarin is F anions whose integral output in the urine was less than the equimolar level of the excreted (IMPA+EMPA+IPA+EtOH). (4) Other corroborative findings were low lethality : of more than 5, 510 patients treated, 11 were acutely dead. (5) Nine exposed males had higher sister chromatid exchange (SCE) rate (5.00±1.48/cell) than the control (3.81±0.697/cell), because dialkyl methylphosphonates seemed to have alkylating activity and producing DNA adducts. The SCE rate also increased after the in vitro exposure of lymphocytes to dialkyl methylphosphonates.

View full abstract

Recently several European Toxicological Socienties have established national toxicology registers. Such societies include the British, Dutch, Finnish, French, Hungarian, and Swiss societies of toxicology. The basis for registration in these societies is peer review evaluation of the applicants. A key-criterion for acceptance is theoretical training covering the main areas of toxicology. Furthermore, job experience is required. After reviewing the merits of the applicant, the register then accepts the applicant to the register usually for five years. As a consequence of this development, EUROTOX, European Societies, of Toxicology, has established a European Register of Toxicologists which will be officially inaugurated during 1997. The register already contains more than hundred British, Dutch, Finnish, and German toxicologists. The European Register of Toxicologists already has had an impact on the recognition of toxicology in Europe, and will strive to promote toxicology education and professional status in other parts of the world.

View full abstract

The PCC-Taiwan was founded in July 1985 under the auspices of the Department of Health, Executive Yuan, and the Veterans General Hospital-Taipei, Republic of China. It has served a population of 21 million inhabitants on a 24-hours basis. It has also served as a referral center for treating poisoning cases nationwide, a training center for physicians and consultants, and a center for Analytical Toxicology. The average annual volume of telephone inquires to PCC is more than four thousand in recent few years and continue to increase annually. The present and future prospective of the PCC-Taiwan which have to be accomplished are:1.to propagate public education of poisoning prevention and increase the utility of PCC before events of intoxication, 2.to establish, computerize and improve the database and network of domestic poisonous products or natural toxins, including herbs, 3.to establish an nationawide referral network for severely poisoned patioents or cluster poisoning events, 4.to build up a global collaborative work with other poison centers.

View full abstract