Assessment of accumulated body burden of metals.

Knowledge of the body burden of a metal is important for evaluation of exposure and risk. Traditionally, the burden has been estimated through levels in blood, urine, hair, or shed teeth, or by mobilization tests. However, all these methods have limitations. In vivo methods for determination of cadmium in kidney by neutron activation analysis or X-ray fluorescence (XRF) reflect the burden, long-term exposure, and risk of toxic effect. In vivo determination by XRF of lead in fingerbone, tibia, or calcaneus reflect the long-term exposure, and should become a valuable tool in epidemiological studies, especially of chronic effects.


Classification of chemicals for carcinogenic and mutagenic properties.

In the framework of the EC labelling guide, three categories were devised for carcinogenic as well as for mutagenic chemicals. In short, category 1 is for compounds shown to produce these effects in man, whereas category 2 is meant to contain substances that should be regarded as such, generally on the basis of sufficient experimental data. Category 3 is for compounds that cause concern but for which the evidence is not sufficient for 1 or 2. These general statements have been implemented, both for carcinogenicity and mutagenicity, by a set of criteria and considerations of the group of "specialized experts" who are supporting the "national experts" in deciding upon the classification of individual compounds. Essentially, this classification relies on the strength of the experimental or epidemiological evidence rather than on considerations of risk. In other words, it is based on proven intrinsic properties of the substances. Compared to the IARC classification for carcinogens, the EC criteria rely more on mechanistic data and on qualitative considerations concerning the relevance for man, and less on a strict count of the amount of positive experimental evidence. Difficulties of this (and any) classification system include: (1) the problem of classifying with limited data and (2) the fundamental problem involved in forcing sets of multi-facetted, and often unsatisfactory, scientific data into a simple one-dimensional scheme that may have huge economic consequences.


The European Community classification of chemicals for reproductive toxicity.

The classification and labelling of dangerous substances was first introduced in 1967 in the European Community with Council Directive 67/548/EEC known as the Dangerous Substances Directive. The "6th Amendment" to this directive in 1979 introduced a notification procedure and a requirement for labelling chemicals for toxicity. Three special categories for labelling were for "Carcinogenicity, Mutagenicity and Teratogenicity". The teratogenicity classification was restricted to chemicals inducing "teratogenic" effects in the classical sense of the word ie. producing only gross structural malformations. Discussions by expert advisors to the European Commission over several years has lead to a widening of concern in this area of toxicology and under the forthcoming "7th Amendment" the classification of "Teratology" will be changed to "Toxic to Reproduction". This will include adverse effects on fertility, pre- and postnatal development and lactation and will encompass not only structural but also functional deficits. This will bring about a major change in the testing requirements to allow adequate classification of chemicals for these other aspects of reproductive toxicity.


Public health problems of organic solvents.

Selected topics of public health importance in toxicology of organic solvents are reviewed. Organic solvents are commonly used as mixtures rather than individual solvents, except for the case of degreasers. Nevertheless, toxicity of mixtures remain mostly to be studied. Among the solvents in general, toluene is apparently the most popular. Narcotic effects are common with all solvents (independent of chemical structure) at high concentrations, and result in an increased incidence of various CNS-related subjective symptoms at concentrations in excess of current occupational exposure limits. Chronic toxicity, teratogenicity and carcinogenicity seems to be related to a given chemical structure. Among the recently reported effects are blindness of "sniffers" by methanol inhalation and teratogenicity of ethylene glycol derivatives in experimental animals. In environmental health, pollution of ground water as well as the general atmosphere by chlorinated hydrocarbons has provoked serious public concern. In addition, emission of certain chemicals including chlorofluorocarbons is recognized to deplete ozone in stratosphere, which may result in human health effects.


Methodology in neurotoxicology--activities within the World Health Organization and International Programme on Chemical Safety.

For many years, the potential deleterious effects of environmental factors on the human nervous system has been examined by several programmes within the World Health Organization (WHO). This presentation will concentrate on the development of methodology to determine the health risks from chemical exposures both in the work place, as well as, the general environment. IPCS as a scientifically based cooperative programme of the United Nations Environment Programme, International Labour Organisation, and WHO, has as one of its goals the development and use of methods to assess human health and environmental risks from chemicals. In this presentation, emphasis will be given to the development by IPCS of an integrated multidisciplinary approach for assessing the neurotoxic potential of chemicals and the risks to human health. The complexity of the nervous system and the variety of effects caused by chemicals will demand several tests in a multidisciplinary approach if the neurotoxic risk of any chemical is to be adequately characterized. Efforts by IPCS to evaluate the use of in vitro and neurobehavioural tests for screening purposes will be discussed as will the roles played by pathology, biochemistry, and neurophysiology in the characterization of neurotoxic risk. Finally, the WHO neurobehavioural core battery developed within the Occupational Health Programme will be described briefly.


Principles of identifying and characterizing neurotoxicity.

There is currently considerable interest in the neurotoxic effects of environmental pollutants. Some of this interest is due to epidemiological, clinical and laboratory studies showing that the nervous system is a target for many toxic substances. The interest is also due to a realization of how little is actually known about the neurotoxicity of most environmental pollutants. Laboratory research in neurotoxicology can be viewed as having two distinct approaches that focus on either the identification or the characterization of neurotoxic substances. Research on the identification of neurotoxicity deals mainly with the screening of chemicals for neurotoxicity. There has been a long-standing tradition of screening chemicals for neurotoxicity. There have, however, been several recent developments that are likely to improve our ability to identify neurotoxic substances, including more detailed assessments of a variety of behavioral and neurological functions. Research on the characterization of neurotoxicity deals mainly with efforts to discover the mechanism(s) of action of neurotoxic substances. This type of research has in some cases significantly advanced our knowledge of neurotoxic effects (e.g., acrylamide, n-hexane). Characterization-based research addresses many of the extrapolation issues of concern in toxicology (e.g., acute to chronic, high-dose to low-dose), and specifically attempts to interrelate the cellular, molecular and functional (neurophysiological, neurobehavioral) effects of toxic substances. These two research approaches represent critical elements of a tiered testing approach that could ultimately lead to more efficient testing protocols and a more comprehensive understanding of pollutant-induced neurotoxic risk in human populations.


International validation of a neurobehavioral screening battery: the IPCS/WHO collaborative study.

A neurobehavioral screening battery consisting of a functional observational battery (FOB) and an automated measure of motor activity is the subject of an international collaborative study. Eight laboratories (four in Europe, four in the U.S.) are participating in this study, which is sponsored by the International Programme on Chemical Safety within the World Health Organization. Representatives from each laboratory received training on testing procedures and the study protocol during a 2-day workshop. Each laboratory then conducted studies using positive control chemicals to demonstrate their proficiency with the techniques. For motor activity studies, each laboratory had to show acute increases and decreases in activity produced by triadimefon and chlorpromazine, respectively. Using the FOB, each laboratory had to detect certain neurological syndromes: tremorigenic activity of a single dose of p,p'-DDT, cholinergic signs with parathion, and neuromuscular deficits with short-term (1-2 weeks) repeated administration of acrylamide. In the formal studies, the effects of seven chemicals are currently being determined following both acute and 4-week exposures. The chemicals include triethyl tin, acrylamide, parathion, p,p'-DDT, toluene, lead acetate, and N,N'-methylene bisacrylamide. All chemicals were provided to the laboratories from a single supplier. Each laboratory is conducting the studies under their standard conditions, using their own strain of rat and testing equipment. Each laboratory also determines a maximum-tolerated dose for each compound as well as the time of peak effect following acute exposure. A simple algorithm is then used to select doses for the formal acute and repeated-exposure experiments. These studies will provide information regarding the reliability and robustness of neurobehavioral screening methods over a wide range of laboratory conditions.


Integration of behavioral and neurophysiological approaches in neurotoxicology.

The validity of behavioral and neurophysiological models can only be assessed with respect to the type of the modelled effect: for acute changes in activation level as well as for chronic motor or sensory deficits both approaches are equally prone to misinterpretation. Validation of behavioral criteria supported by neurophysiological correlates and vice versa is the best protection. In the cognitive and emotional sphere, a shift to testing of the readiness ("fluidity") dimension seems to be promising both in human and animal studies, permitting also more reliable extrapolations and efficient cooperation of behavioral and neurophysiological approaches.


Developmental aspects of neurobehavioural toxicity.

Previous work on the developmental aspects of neurobehavioural toxicity in rats and mice has shown the reliability of a variety of procedures aimed at assessing changes that may have widespread functional consequences, for example: (i) modified Fox batteries to study the maturation of various reflexes and responses after birth, (ii) activity/habituation and analgesia tests with age-specific profiles of reactivity to selected drug challenges, and (iii) simple learning tasks such as active and passive avoidance [1]. We will now summarize more recent work on other portions of the behavioural repertoire which deserve to be thoroughly assessed in "higher-tier" studies.


Extrapolation from animals to humans: scientific and regulatory aspects.

The necessity to protect humans from the adverse effects of chemicals on structure and function of the developing and/or mature nervous system is increasingly recognized among regulatory bodies throughout the world. Whereas structural changes dominated much of neurotoxicological research in the past, functional markers of neurotoxicity are gaining acceptance as early signs of insult. Among the more ambitious protective efforts are legal requirements of premarket testing for "behaviour-disrupting properties" of chemicals. Assumed or proven validity of cross species extrapolation underlies the use of animals in primary or secondary screening schemes. Two steps must be distinguished here. The first step is endpoint-base or qualitative, whereas the second one is dose-based or quantitative. Species comparisons in terms of endpoints is typically done within a framework of broad functional categories, such as sensory, motivational, cognitive, motor or social functions. Dose-based extrapolation requires knowledge about species differences in terms of toxicokinetics or metabolism in order to arrive at valid translations of dose-response contingencies. Principles of cross-species extrapolation in neurotoxicology will be exemplified by means of representative neurobehavioural and neurophysiological findings for neurotoxic chemicals of environmental concern, for which an adequate data base is available for comparative purposes, such as inorganic lead and polychlorinated biphenyls (PCB).


Molecular biology approaches to biological monitoring of genotoxic substances.

Genetic testing is subdivided into genetic monitoring (evaluation over time of induced genetic changes) and genetic screening (detection of inherited traits). Genetic factors in relation to susceptibility to environmental agents are briefly examined, as well as mutation assays suitable for use in genetic monitoring, techniques for identifying specific DNA lesions, and oncogene products as biomarkers. In vitro studies with AS52 Chinese hamster ovary cells indicate that the distribution of lesions (e.g., point mutations or segment deletions) at the xanthine-guanine phosphoribosyl transferase (gpt) gene in mutants generated by exposure to nickel compounds show some substance specificity. This ability is viewed as a promising development for the molecular epidemiology of occupational and environmental cancers. It is concluded that technical limitations pertaining to specificity and sensitivity, as well as ethical and legal implications, need to be resolved before routine application of genetic monitoring and screening is feasible.


Pre-hospital strategy for therapeutic intervention of fire victims.

Fire victims are exposed to the triple threat of thermal injury (skin and lungs), smoke toxicity (toxic or irritant gases and soots) and even trauma whose occurrences are somewhat independent one from the other but whose addition does sharply increase the probability of death of the victims. As the different victims of the same fire may not suffer from the same injuries, this triple threat must be looked for in each fire victim.


Hospital treatment of victims exposed to combustion products.

Combustion toxicology is complex so, although victims exposed to combustion products are mainly treated symptomatically, it is important to identify those situations when specific therapeutic measures might be of importance. Victims presenting respiratory symptoms including severe cough, bronchoconstriction, hypoxia and respiratory distress should be given oxygen and ventilatory assistance or support. Furthermore, bronchoconstriction should be treated with bronchodilators (beta-2-adrenoreceptor agonists, theophylline). Corticosteroids should be considered both for inhalation and systemically due to the risk of developing toxic pulmonary oedema that may appear after a symptom-free interval that might last up to 48-72 h. Victims with impaired consciousness should be regarded as being exposed to carbon monoxide and cyanides. Apart from oxygen and optimal symptomatic treatment hyperbaric oxygen therapy should be considered in carbon monoxide poisoning. Certain cyanide antidotes, namely those with low intrinsic toxicity (as sodium thiosulphate, hydroxocobalamin) should be given liberally in these situations. Other specific therapeutic measures that might be considered when appropriate are administration of organophosphate antidotes (atropine, oximes), heavy metal chelators (e.g. dimercaptopropane sulfonate, dimercaptosuccinic acid) and methemoglobinemia antidotes (methylthionine, toluidine blue). Inhalation of hot fumes may cause upper respiratory tract oedema (e.g. laryngeal oedema) necessitating orotracheal intubation and ventilatory support.


Progress and challenges in the preclinical assessment of cytokines.

This presentation focuses on the history and status of the preclinical toxicologic assessment of recombinant proteins. Cytokines and growth factors are used as examples. There has been an evolution of thought over the past dozen years on the testing of these substances that has ranged from their being considered nontoxic, human-specific proteins for which no predictive testing could be done in animals, to the present view that they can be toxic, relevant testing may be possible in animals, and assessment approaches should be science-based and case-by-case. The challenge of appropriate testing of recombinant proteins has caused toxicologists in both industry and in regulatory authorities to reconsider not only testing procedures but the purpose of preclinical assessment. The form of regulatory guidelines (guidance vs. inflexible testing protocols) has been questioned, and the need for interaction between scientists in industry and regulatory agencies has been strengthened. This process has advanced the science of toxicology.


Preclinical evaluation of recombinant human interleukin-4.

Recombinant human IL-4 (rhuIL-4) is primate-specific and produces multiple biologic effects on lymphoid cells involved in protection against cancer. RhuIL-4 was evaluated in the cynomolgus monkey to support clinical studies for the immunotherapy of cancer. Administration of rhuIL-4 to monkeys by SC injection of 0, 0.5, 2.5 or 12.5 micrograms/kg BID for one-month (with two-week recovery) resulted in alterations in clinical chemistry and hematology (CCH) parameters consistent with a consumptive coagulopathy. Histomorphologic evaluation revealed increased granulopoiesis, testicular atrophy, and proliferative and inflammatory vascular lesions (VL). IVL principally affected the arterial tree with some proliferation of medial smooth muscle. During the latter part of the treatment and recovery period. CCH parameters approached or returned to pretreatment values, the former finding attributed to the production of antibody to rhuIL-4. At final necropsy, bone marrow appeared normal, and IVL decreased in incidence and severity. ELISA studies of serum indicated 50-90% of the monkeys developed antibody titers > 1000 by Day 22 (not observed in man). The frequency and severity of adverse effects due to rhuIL-4 in the clinic appear to be does-related and reversible with few objective responses to therapy observed. Common toxicities included milk to moderated fever and fatigue and an occasional change in hematopoietic, hepatic and renal function. The monkey predicted hematologic findings, but not all target organ effects.


Pathology induced by interleukin-6.

Interleukin 6 (IL-6) is a multi-functional cytokine which plays an important role in the immune response, hemopoiesis and host defense. Recombinant human IL-6 (rhIL-6) was administered at high doses to mice, rats and non-human primates. In all species IL-6 had an immunostimulatory and hemopoietic (especially on megakaryocytes) effect. An acute phase response was most pronounced in non-human primates, which was not, however, associated with any major histopathological liver change. Finally, no evidence of glomerular pathology was found. Neutralising antibodies were detected within 10 days of rhIL-6 administration in all species.


Utilization of homologous proteins to evaluate the safety of recombinant human proteins--case study: recombinant human interferon-gamma (rhIFN-gamma).

Interferon-gamma is an immunomodulatory cytokine that has an extremely restricted host range of activities. RhIFN-gamma was one of the first species-specific recombinant proteins to be assessed in conventional safety models typically utilized for xenobiotics. Acute, subchronic and Segment I and II reproductive studies in rats revealed no evidence of toxicity at any of the doses tested; these results were not predictive of clinical toxicity, which is not unexpected since rodents are known to be pharmacologically nonresponsive to rhIFN-gamma. In contrast, 4- and 13-week multidose toxicity studies in cynomolgus monkeys with rhIFN- were predictive of many of the dose-limiting clinical toxicities. RhIFN- is active on non-human primate cells, though not at the same level as on human cells. In addition, qualitative similarities were observed between toxicity studies employing rhIFN-gamma in the cynomolgus monkey and recombinant murine interferon-gamma (rmuIFN-gamma) in the mouse. These results suggest that in situations where a high degree of species specificity is encountered, studies employing a recombinant protein in a homologous species may provide a useful test system for preclinical safety assessment. This information should be evaluated in conjunction with data from studies conducted with the human protein in pharmacologically responsive animal models when possible.


Experience with the preclinical assessment of basic fibroblast growth factor (bFGF).

Repeated intravenous administrations were carried out in cynomolgus monkeys and rats (S.D.) for a maximum of 4 weeks at doses of 1, 10 and 100 micrograms/kg/day in stable formulation. Three main target organs were identified: red blood cells (RBC), kidney glomeruli (KG) and bone at the top dose level. RBC: Normochromic normocytic anaemia started in rats and monkeys during the second week of treatment (decrease in red blood cell production). The kinetics of this anaemia, as well as its recovery, will be discussed. Bone: Dramatic hyperostosis in rats was present by day 10 in long or spongious bone. This became marked on day 29 and regressed after treatment was stopped. KG: In the rat glomerular lesions were present starting from day 16. They consisted of enlargement and vacuolation of podocytes with loss of foot processes and adhesions between glomerular tuft and Bowman's capsule. Proteinuria was a striking feature. In the monkey the lesions were hyperplasia of the parietal epithelium of Bowman's capsule which involved replacement of normally flattened epithelium by cuboidal cells, with some pseudostratification. Proteinuria also occurred in monkeys, accompanied by a lowering of serum protein (albumin). In two animals, death (by day 15) was preceded by high levels of urea and blood creatinine. The above lesions (KG) disappeared almost completely over a recovery period. It is suggested that these phenomena are not the expression of direct toxicity in the form of lethal insults, but rather a manifestation of a change in cell activity.


Monitoring of early nephrotoxic effects of industrial chemicals.

Long-term exposure to certain industrial chemicals (e.g. heavy metals, some halogenated hydrocarbons) may cause progressive degenerative changes in the kidney, possibly leading to renal insufficiency. The screening tests most widely used to assess the integrity of the kidney (i.e. serum creatinine or BUN and the quantitative or semi-quantitative measurement of total proteinuria) lack sensitivity; they do not permit the detection of renal disturbances at a stage when removal from exposure may prevent progression of the disease process and are not suitable to determine the no-effect levels of potentially nephrotoxic chemicals. During the last decades new markers have been proposed for the early detection of structural and/or functional changes at various sites of the renal parenchyma. Some tests mainly attempt to assess the integrity of the glomerulus (e.g. high Mr proteinuria such as transferrinuria and albuminuria; increased excretion of some components of the glomerular basement membrane or the mesangium matrix, increased plasma concentration of low Mr proteins such as beta 2-microglobulin and free retinol binding protein), the proximal tubule (e.g. urinary excretion of several low Mr plasma proteins, tubular enzymes and antigens), the loop of Henle and distal tubule (e.g. excretion of various prostanoids). Currently, the majority of these tests are of limited value at the individual level because their health significance, even when they are persistently abnormal, has not yet been sufficiently studied. In workers exposed to Cd, however, it has been shown that a persistent low Mr proteinuria is predictive of an exacerbation of the age-related decline of the GFR; this biological change should be considered as an adverse effect. Currently, the principal application of these tests lies in the framework of epidemiologic studies designed to assess permissible exposure levels to nephrotoxic pollutants. The study of dose-effects/response relationships based on a large battery of renal markers has allowed the better determination of the internal dose of Cd, which is not associated with significant renal risk.


Current guidelines for the preclinical safety assessment of therapeutic proteins.

Guidelines concerning the preclinical safety assessment of therapeutic proteins are available, e.g. in the European Community (EC Notes for Guidance). Unlike rather rigidly prescribing test systems such as those known for chemical substances, it is agreed that for biotechnologically-produced products, testing may be quite different. Those guidelines which allow for a true case-by-case development have been considered the best; however, regulatory authorities are expected to disagree ex post with the approach chosen earlier. To allow for a scientific rationale, which includes the freedom to deviate from the expected norm, guidance must include an offer for discussion of requirements. This combination of recommendations (not requirements) with optional discussions has been the European approach. Although the American approach is known to be somewhat more case-by-case oriented, and the Japanese approach is known to be somewhat more strict, the outcome of drug development, i.e. the application of guidance, in the three regions has been strikingly similar. This underlines the importance of a pharmaceutical manufacturer and his ability to interpret the rules in the light of the product to be developed.


Difficulties in conceiving and applying guidelines for the safety evaluation of biotechnologically-produced drugs: some examples.

Pharmaceutical companies and regulatory authorities need guidelines for the safety evaluation of all drugs. Nevertheless, new types of drugs, e.g. biological products produced by novel biotechnological processes (as recombinant DNA methods), induce new problems for this evaluation. The following points will be assessed: (1) As a postulate, safety is highly dependent on the industrial process and on the controls. (2) To conceive guidelines, it is necessary to take into account the structure of the pharmaceutical product compared with the natural biological product (identical or not), the therapeutical use and the relevance of the classical toxicological investigations: for example single dose toxicity gives poor results for target organs, repeated dose toxicity is related to immunogenicity, mutagenicity testing is debatable. (3) To apply guidelines is still more difficult, for example to select animal models (animal models of diseases), define dose levels, evaluate immunogenicity, conceive the design of reproduction studies and the feasibility of cancerogenicity studies. Therefore, common sense and a case-by-case approach are necessary (guidance vs. guidelines) for the safety evaluation of drugs produced by biotechnology, and an early collaboration between all the partners (companies, experts and regulatory authorities).


Structural and functional aspects of the thyroid follicular epithelium.

The thyroid epithelium is morphologically and functionally polarized, with an apical surface facing the follicular lumen containing colloid and a basolateral surface facing the interstitium. Iodination and thyroid hormone synthesis occur in the colloid at the apical plasma membrane. The introduction by Mauchamp et al. of primary cultures of porcine thyroid cells grown as a polarized, confluent monolayer on a filter in a bicameral chamber system has now made it possible to study in more detail the barrier function and vectorial ion transport in the thyroid epithelium. The follicular cells form a very tight monolayer (transepithelial resistance > 6000 ohm cm2) and establish a transepithelial potential difference (apical medium negative) of about 20 mV. These parameters are rapidly influenced by TSH, mainly by an action on apical sodium channels, and by EGF. The integrity of the barrier is, as in other epithelia, dependent on extracellular calcium. A calcium-dependent cell adhesion molecule, uvomorulin, is expressed at the lateral plasma membrane surface. EGF induces cell proliferation as well as migration of some of the epithelial cells to a position below the monolayer, which however maintains its polarity and barrier function. In contrast, during TPA-induced proliferation the barrier function is disrupted. Iodide is vectorially transported in basoapical direction while the epithelial layer is virtually impermeable for iodide transfer in the opposite direction. Iodide is concentrated in the cell by the basolateral "iodide-pump" and its efflux across the apical plasma membrane is rapidly and selectively increased by TSH via cAMP. EGF inhibits vectorial basoapical iodide transport mainly by reducing the iodide permeability of the apical plasma membrane. Together, these recent observations indicate that the ion content of the follicular lumen is strictly controlled by the thyroid epithelium.


Cell proliferation and thyroid neoplasia.

The classic experimental model of thyroid neoplasia using radiation as a mutagen followed by long-term goitrogen treatment gives rise to multiple tumours in rats. Radiation alone, with suppression of TSH produces no tumours, TSH-induced growth alone causes a low level of tumorigenesis. Cell proliferation in this model is therefore critical. Epimutation as well as mutation is important. Rodent and human thyroid tumours show a clear stepwise progression, associated with both morphological and oncogene changes. In experimental animals the finding that monoclonal adenomas and carcinomas induced in the presence of long-term high TSH retain TSH dependency suggests that the step in the tumour progression requiring the development of TSH independent growth is bypassed, explaining the frequency of tumour development in this model. Normal thyroid follicular cell growth is limited, and a genotoxic effect before the growth plateau is more effective in carcinogenesis than a genotoxic effect after a period of growth. These observations will be interpreted in relation to the importance of thyroid tumours in regulatory toxicology and the pathobiology of thyroid tumours in man.


Chemical carcinogenesis in the thyroid gland.

Chemical carcinogens and promoters in thyroid carcinogenesis may be divided into two types, one having goitrogenic effects, the other not having these effects e.g. phenobarbital, 4,4'-diaminodiphenylmethane. The influence of sex hormones like estrogen and testosterone remains unclear, although some thyroid tumors have estrogen receptors. Further studies in this area are necessary.


Individual susceptibility to toxicity.

Individual variation in susceptibility to chemical toxicity may be due to differences in toxicokinetic patterns or effect modification. Well-documented interspecies genetic differences in susceptibility to chemicals had lead to studies of such variation also within species. Epidemiological evidence now suggests that common variations, particularly in the P-450 enzymes, may play a major role in determining individual susceptibility to chemically-induced disease. Physiologic factors are involved in the particular susceptibility of the fetus, the newborn, and the old. Constitutional susceptibility is also affected by acquired conditions, including chronic disease, such as diabetes mellitus. Perhaps the most complex area relates to the increase in vulnerability caused by previous or contemporary exposure to other factors, thus eliciting, e.g., synergistic effects. Although amply demonstrated by experimental studies, epidemiological or clinical confirmation is generally lacking. One hypothesis suggests that a chemical exposure may affect the reserve capacity of the body, though not resulting in any immediate adverse effect. Subsequently, the body becomes unable to compensate for an additional stress, and toxicity then develops. Epidemiological approaches are available and need to be expanded. Research in this area has potential ethical implications which should be dealt with in an open, informed forum.


Second messengers in cholinergic-induced convulsions and neuronal injury.

Acetylcholine (ACh) is a powerful excitatory neurotransmitter in the brain. Stimulation of brain cholinergic muscarinic receptors (mAChR) cause persistent tonic-clonic convulsions. mAChRs are coupled to G-protein which mediates the receptor stimulation to phospholipidase C (PLC). PLC hydrolyses phosphatidylinositol-4,5-bisphosphate (PI), a membrane phospholipid, into two second messengers, inositol-1,4,5-trisphosphate (Ins(1,4,5)P3), and diacylglycerol (DAG). Both messengers cause neuronal stimulation and when in excess, may contribute to neuronal injury. Indirect cholinergic agonists organophosphates (OPs) such as soman, paraoxon, and malaoxon, and direct cholinergic agonists, such as pilocarpine, are powerful convulsants. They stimulate brain mAChR-coupled to PI signalling as indicated by decreased brain inositol and increased brain inositol monophosphates, metabolites in PI turnover, and indirectly reflect the activity of the brain PI system. In rats, during cholinergic convulsions, brain inositol decreases, and inositol monophosphates increase prior to and during convulsions. Persistent convulsions cause neuronal injury especially in the hippocampus and cortex, and associated increase in brain Ca2+. The mechanisms of convulsions and associated neuronal have remained open, but both in vitro and in vivo data provide evidence that facilitated PI signalling and increases in free intracellular Ca2+ may have an important role in these events. Age and female sex amplify the effects of cholinergic brain stimulation and convulsions.


Field studies of aflatoxin exposure, metabolism and induction of genetic alterations in relation to HBV infection and hepatocellular carcinoma in The Gambia and Thailand.

The relative contribution of aflatoxins (AF) and hepatitis B virus (HBV) to the aetiology of liver cancer remains to be determined, as does the mechanism of any interaction between these two factors. Methods to measure individual exposure to AF permit the assessment of this possible interaction in field studies. The measurement of AF covalently bound to albumin in peripheral blood has been particularly useful in this respect. In east and west African countries the majority (75-100%) of individuals has been found positive (> 5 pg AFB1-lysine eq./mg albumin) for the AF-albumin adduct with levels ranging up to 720 pg/mg. Levels of adduct to date have been age- and sex-independent, although marked seasonal variations were seen in The Gambia. Exposure also occurs in utero, with the AF-adduct being found in umbilical cord blood. In a study in The Gambia involving 323 children (age 3-8 years) the AF-albumin adduct levels were examined with respect to HBV infection and ethnic group. Over 95% of all sera contained detectable adduct but children positive for HBV surface antigen (HBsAG) had significantly higher adduct levels than children with markers of past infection or who had never been infected (mean (log) AF-albumin adduct levels 4.41 +/- 0.95, 4.04 +/- 0.99, and 4.05 +/- 1.03 respectively, p = 0.04). In addition, there were highly significant differences in adduct levels between the three major ethnic groups (Wollof 4.41 +/- 0.69: Fula 4.05 +/- 1.1; Mandinka 3.7 +/- 1.14). Wollof children were also more likely to be HBsAg positive than the other two groups. These data suggest that ethnic group and HBV infection can influence AF metabolism and this is being examined in this population with respect to genetic polymorphisms in cytochrome P450 and glutathione-S-transferase enzymes. In addition, these biomarkers are being compared to the nature and frequency of mutations in somatic and tumour cells.


The use of biomarkers in epidemiology: the example of bladder cancer.

Epidemiological studies have suggested that genetically based polymorphism for N-acetylation of arylamines might play an important role in the susceptibility to bladder cancer induction. However, these studies show large differences in the extent of such susceptibility. We have undertaken collaborative investigations (with IARC, MIT, NCI and NCTR) which couple internal dosimetry among smokers (measurement of hemoglobin and DNA adducts of arylamines) with the assessment of metabolic polymorphism. In one of these studies, hemoglobin adducts of 14 arylamines (including 2-naphthylamine and 4-aminobiphenyl) were analysed in a group of 86 subjects (smokers and non-smokers) in order to establish whether the inter-individual variability left unexplained by tobacco smoking could be attributed to differences in individual metabolic patterns. In another investigation on 100 smokers and non-smokers, metabolic polymorphism for N-acetylatransferase was assessed by measuring five different urinary metabolites of caffeine, after timed urine collection following coffee consumption. Arylamine-hemoglobin adducts were also measured. 4-Aminobiphenyl-hemoglobin adducts were found to be related to both the quantity and the type of tobacco smoked, as well as to the acetylator phenotype (independently of smoking habits).


Advantages and limitations of laboratory methods for measurement of carcinogen-DNA adducts for epidemiological studies.

In molecular epidemiological studies, the measurement of carcinogen-DNA adducts in human tissues can provide direct evidence of current exposure to chemical carcinogens. Moreover, data on steady state DNA adduct levels and the rate of cell proliferation can be related not only to carcinogen-target tissue dosimetry but may also be useful in assessment of human cancer risk. Thus far, laboratory methods for adduct detection have primarily utilized 32P-postlabelling, immunoassays, and mass spectrometry. However, accurate quantitation of DNA adducts requires knowledge of the structural identity and chemical properties of carcinogen-base adducts, the availability of synthetic standards for recovery determinations, and the development of complementary methods to corroborate analytical findings.


Expression of pulmonary cytochrome P4501A1 and carcinogen DNA adduct formation in high risk subjects for tobacco-related lung cancer.

Cigarette smoking is the strongest risk factor for lung cancer (LC), but genetically determined variations in pulmonary metabolism of tobacco-derived carcinogens may affect individual risk. Results from a case-control study on LC patients demonstrated the pronounced effect of tobacco smoke on pulmonary xenobiotic metabolism and prooxidant state, and suggested the existence of a metabolic phenotype at higher risk for tobacco-associated LC: LC patients who were recent smokers had significantly induced BP-3-hydroxylase (AHH) and ethoxycoumarin O-deethylase (ECDE) activities in lung parenchyma, when compared with smoking non-cancer patients. In recent smokers, lung AHH activity was positively correlated with the level of tobacco smoke-derived DNA adducts as determined by 32P-postlabelling. Pulmonary AHH activity also showed a good correlation with the intensity of immunohistochemical staining for cyt. P4501A by a monoclonal Ab in lung tissue sections: smoking and peripheral type of lung cancers were positively related to high levels of this cyt. P450 species, probably reflecting high rates of induction. These results suggest that high pulmonary CYP1A1 expression (controlling in part carcinogen DNA-adduct formation) in tobacco smokers, appears to be associated with LC risk. High risk subjects may thus be identifiable through genotyping assays for CYP1A1 polymorphism.


Use of biomarkers in epidemiology: quantitative aspects.

Cancer initiators (mutagens) present, due to the absence of definable no effect threshold, a special problem in toxicology, requiring a high sensitivity of detection methods. Disease epidemiology aiming at identification of carcinogens and quantification of associated risks has a low resolving power, the detectable incidence or mortality increments being often orders of magnitude larger than those which are of public concern. Other drawbacks of disease epidemiology is the long latency times and the influence of confounders. The use of genetic endpoints as biomarkers suffers from low cause specificity, although this drawback seems to be overcome, partly at least, by emerging methods for determination of mutation spectra at the DNA level. Proximal cancer initiators/mutagens are electrophilic compounds or metabolites that can react with nucleophilic atoms in nucleic acids and proteins. These reactions lead to 'adducts' that can be identified and quantified, e.g. in lymphocytes and erythrocytes in blood samples. The shift from biological observations to chemical analysis permits sufficient sensitivity, and measurement can be done shortly after onset of exposure. The well-defined life span of the adducts to hemoglobin (Hb) offer possibilities of dose calculation and risk estimation. For these reasons the measurement of adducts to Hb and DNA constitutes a powerful epidemiological tool, applications of which has been initiated in work environments and the general environment and also in the search for a priori unknown carcinogens.


Hazard assessment of chemical contaminants in soil.

Disposal practices, accidental spills, leakages and local aerial deposition occurring in the past have led to local soil pollution in many cases. Especially in situations where people live on or nearby such locations this has created concern about possible adverse effects on human health. A stepped approach to the hazard assessment of polluted soil, as developed by a Task Force from the European Chemical Industry Ecology and Toxicology Centre (ECETOC), is described. In an early phase in the assessment process the potential exposure of humans is estimated. The Human Exposure to Soil Pollutants (HESP) model can be applied for this purpose. The model calculates the total exposure of adults and children resulting from pollutants present in soil, via 10 different exposure routes. The estimated exposure can be used to indicate the potential significant exposure routes and to carry out a preliminary hazard assessment. The model is also able to predict pollutant concentrations in soil which do not exceed accepted maximum exposure levels for humans in both standardised and site specific situations. The stepped approach is cost-effective and provides an objective basis for decisions and priority setting.


Hazard assessment in freshwater ecosystems.

Hazard assessment of chemicals in freshwater environments depends on comparing concentrations that are expected to occur in water and sediment, i.e. expected environmental concentrations (EEC), with those that are estimated to have no biological effects, i.e. the no-observed effect concentrations (NOEC). The difference between these two estimates is the margin of safety. The EEC can be estimated from data for chemical release rates, physicochemical properties and environmental parameters that affect transport and transformation. The NOEC can be estimated from the results of toxicity tests using aquatic plants, invertebrates and fish. When making these estimates it may be necessary to extrapolate from relatively limited laboratory data to the real world. Inevitably, this involves some degree of uncertainty. Such uncertainty can often be resolved by carrying out controlled field tests, using small, outdoor enclosures (microcosms), relatively large, outdoor ponds (mesocosms) and experimental streams. In this paper the advantages and disadvantages of various experimental approaches and systems will be reviewed.


Advances in prediction of exposure for organic chemicals.

In the late 1970s new legislation was set up in industrialized countries to protect the environment. This has led to the definition of a new branch of science: ecotoxicology. The main objective of applied ecotoxicological research is the elucidation of indications of toxicity to be utilized for risk assessment and hazard evaluation for the management of potentially harmful chemicals. The development of predictive approaches for the hazard assessment of chemical substances has given an increased relevance to exposure evaluation. This paper discusses methods and processes used to estimate and assess the environmental distribution and fate of chemicals for the ultimate purpose of determining exposure of the general population via soil, water, food or air. Parameters useful for the assessment of risk for different time and space scales are discussed. A collaboration between toxicologists and ecotoxicologists is proposed to improve the management of chemical substances.


Biochemical responses as indicators of toxic effects of chemicals in ecosystems.

Biochemical responses of animals to environmental chemicals (biochemical biomarkers) can give measures of exposure, and sometimes also toxic effect. They are particularly valuable where they can be used to measure the toxic effects of chemicals in the field, employing non-destructive sampling methods. Measurements of exposure are useful in the case of non-persistent chemicals (e.g. organophosphorus, carbamate, or pyrethroid insecticides) which are difficult or impossible to detect by chemical analysis. They can also be useful to provide an integrated measure of the level of exposure to a group of related chemicals. Biochemical biomarkers are likely to provide a measure of toxic effect, where they are based upon a molecular mechanism which underlies toxicity. A widely-used biochemical biomarker is cholinesterase depression, which may involve destructive sampling (brain acetylcholinesterase) or non-destructive sampling (serum butyrylcholinesterase). For genotoxic chemicals, techniques which measure DNA damage (e.g. detection of DNA adducts) provide a powerful tool in measuring environmental effects. The detection of biochemical changes caused by anticoagulant rodenticides (e.g. abnormal levels of clotting proteins in blood) provides another example of this approach. In general, the development of simple, sensitive, and specific assays that are 'user-friendly' would open the way for much wider use of biochemical biomarkers in environmental monitoring.


Evaluation of adverse effects in the standard-setting process.

Occupational exposure limits (OELs) were first introduced more than a century ago in Germany [1]. They were based on observations of people exposed at the workplace, and on experimental exposures of humans and animals, all accompanied by analytical determination of airborne occupational toxicants. The "acceptable concentrations for short-term and long-term exposure" were derived using crude subjective criteria (humans), or gross pathological alterations (animals). Over the years considerable refinement of these criteria has been achieved, both in their type and number, starting from overt histological derangements, going on to the physiological and biochemical level, and even to subtle psychological parameters. This development has taken place in parallel with, and has been considerably influenced by changes in the definition of health and the perception of effects detrimental to health. Differences in the elementary philosophy of health and in the activities aimed at preventing damage to health in different societies have complicated all the efforts to harmonize standard-setting processes at the international level.


Environmental hazard classification of chemicals.

Information on the environmentally dangerous properties of chemicals is of fundamental importance if the users of chemicals are to take account of the risks to the environment in their choice of chemical products, and to organize the use of chemicals and waste disposal in an environmentally safe manner. In order to assist suppliers of chemicals in setting out their product information and to ensure its consistency, it is important to have rules on classification and labelling. Such rules must be based on criteria for environmental hazard classification. During the 1980s specific classification criteria for the aquatic environment were developed in the Nordic countries and by the Commission of the European Communities. These criteria now form the basis for the regulations on classification and labelling of chemical substances in both the EC and EFTA countries. The regulations entered into force in the EC on 1 July 1992 and will be enforced in the EFTA countries as soon as possible. Besides labelling there are several other areas, e.g. transport regulations, where classification criteria are or will be applied on a national or international scale. International harmonization of criteria at this early stage is highly desirable as it will prevent the development of divergent systems which will require harmonization at a later stage. To this end, the OECD initiated a Clearing House activity on the feasibility of elaborating harmonized approaches to environmental hazard classification. The Clearing House recommended adoption of the current classification criteria, joint development of criteria for other sections of the environment and joint development of procedures for updating and methodologies for classification of chemical preparations. Further international work in this area will be coordinated by IPCS. Harmonization of classification is also a programme area under Chemical Forum with IPCS as coordinator as agreed at UNCED 92.


Mechanisms of cytotoxicity caused by antitumour drugs.

Although the nature of the interaction between a drug or toxin and its target is of critical importance in determining the fate of a cell, we have argued here that the biological outcome of that interaction will also be determined by the nature of cellular events "downstream" of the initial interactions. We suggest that some type of coupling must take place between the formation of a drug-target interaction (the stimulus?) and the response of the cell. That response will depend upon the phenotypically determined repertoire of response open to the cell as well as upon the quantitative and qualitative measures of the events that the drug induces (DNA or protein damage, inhibition of growth etc.). For example we have described how the HL-60 cell appears to respond to low levels of toxins by engaging a programme of terminal differentiation whilst at greater concentrations apoptosis becomes engaged. Consideration of the cellular response to a toxic insult may provide valuable insights into the selective toxicity of agents as well as providing avenues for the discovery of toxins which might be useful in the treatment of cancer.


Thiol modification and cell signalling in chemical toxicity.

Exposure of cells to thiol oxidizing agents can result in the modification of key proteins involved in cell signalling. Such changes have been shown to affect agonist-stimulated phosphoinositide metabolism, activation of protein kinases and intracellular Ca2+ signals, which result in abnormalities in cell metabolisms and growth. Here, we show that moderate levels of oxidants potentiate growth signals and either enhance cell proliferation or facilitate cell differentiation, whereas inhibition of growth signals by higher oxidant concentrations can block cell proliferation and activate programmed cell death (PCD). Finally, a general alteration of multiple signalling pathways associated with increased catabolic reactions results in cell death by necrosis. Our data suggest that oxidant interaction with cell signalling systems may exert opposite effects, depending on the dose, and that oxidative reactions may either mimic growth factor stimulation and stimulate cell proliferation or inhibit growth signals and activate PCD, in the same cell systems.


Programmed cell death and its protective role with particular reference to apoptosis.

Apoptosis is a type of programmed cell death involved in growth control of tissues. It is considered as a cellular suicide functionally opposite to mitosis. It may serve to remove "unwanted" damaged or dangerous, e.g. precancerous, cells. Chemical compounds can interfere with the regulatory network which controls apoptosis and can thereby stimulate or prevent cell death. Both induction or inhibition of apoptosis may result in various diseases such as of the immune system, malformation or tumor development. The protective role of apoptosis against carcinogenesis is described in some detail. Tumor formation seems to occur through several stages, namely initiation, promotion, progression, and involves formation and growth of premalignant cell populations. At least in some model systems initiated cells and premalignant cell populations have been found to exhibit enhanced cell replication, but also enhanced apoptotic activity as compared to the normal tissue. Therefore, initiated cells may be eliminated by apoptosis. Tumor promoters can inhibit apoptosis in putative preneoplastic cells and thereby accelerate tumor development. Furthermore, in hormone-dependent cancers malignant cells may undergo massive apoptosis in response to hormone withdrawal or antihormone treatment. Finally, the regulation of apoptosis will be addressed. Our results suggest that transforming growth factor beta 1, a negative regulator of epithelial tissue growth, is a signal inducing apoptosis of liver cells.


Protection from oxidative damage in mouse liver cells.

Paracetamol toxicity in mouse hepatocytes involved oxidative stress initiated by the formation of NAPQI. This oxidative component of paracetamol injury is associated with the latter stages of the poisoning process. Ebselen, a drug with GSH-peroxidase activity, was effective in ameliorating these oxidative events.


The toxic oil syndrome (TOS, 1981): from the disease towards a toxicological understanding of its chemical aetiology and mechanism.

In Spain early in May 1981, 20,000 people became ill with a severe acute respiratory illness. The eosinophilia and subsequent myalgia, scleroderma and muscle wasting indicated a unique disease entity. Epidemiological evidence linked the disease with the consumption of oils containing "refined" aniline denatured rape seed oil. Ten years after the explosive appearance of this disease (approximately 350 deaths and over 1000 in the chronic phase) the clinical and pathological description is now well established. The aetiological agent(s) in the food oil are unknown and the mechanism(s) of pathogenesis are uncertain. There is no experimental animal model. A new disease, Eosinophilia Myalgia Syndrome (EMS) which appeared late in 1989 in the USA, is due to the consumption of impure 1-tryptophan. There may be similarities between the diseases and the aetiological agents for TOS and EMS: possibilities for future research will be discussed. Underlying the time lag for solution of this problem is a lack of knowledge of the basic biology involved.


Interaction and distinction of genotoxic and non-genotoxic events in carcinogenesis.

Multistage carcinogenesis involves genotoxic as well as non-genotoxic mechanisms. The importance of genotoxic events in human carcinogenesis is apparent from the analysis of tumours: for example, five to six genetic alterations can be found in most malignant colorectal tumours. While such measurable "footprints" (e.g. ras, p53 mutations) can be left in tumours by genotoxic events, non-genotoxic events cannot directly generate them. Thus, the lack of specific indicators of non-genotoxic events in carcinogenesis makes the identification of non-genotoxic carcinogens difficult. It is also important to emphasize that apparent "genotoxic" endpoints (mutations, chromosome aberrations) could be induced by "non-genotoxic" agents through indirect mechanisms (e.g. induced cell proliferation and/or genomic instability, oxidative damage, deamination of 5-methyl cytosine). This emphasizes the need for differentiating "events" from the actual "activities" of chemicals and the difficulty of classification of carcinogens into genotoxic and non-genotoxic. One of the best models for the study of interaction of genotoxic and non-genotoxic mechanisms during carcinogenesis is a two-stage carcinogenesis system using mouse skin, rat liver or cultured cells. Molecular analysis of tumours produced on mouse skin by the classical initiation-promotion protocol indicates that the mutation spectra of oncogenes, e.g. Ha-ras, are determined by initiating (genotoxic) and not by promoting (non-genotoxic) agents. However, since usually no tumours appear without the application of tumour-promoting agents, the manifestation of genotoxic events (Ha-ras mutation) is dependent on the action of non-genotoxic agents. Using a BALB c 3T3 two-stage cell transformation system, we have now succeeded in confirming this and have quantitated the initiation and promotion events. These studies may help us not only in understanding mechanisms of carcinogenesis but also in developing molecular quantitative risk assessment in terms of multistage carcinogenesis.


Prediction of non-genotoxic carcinogenesis.

The distinction between genotoxic and non-genotoxic mechanisms of chemical carcinogenesis is now generally accepted. This acceptance implies a revised approach to the definition and measurement of genotoxicity. It is proposed that genotoxicity should be assessed as part of the overall evaluation of the toxicity of chemicals, not as a separate and isolated activity. The level and duration of dosing plays a critical role in the observation of genotoxic phenomena, and such parameters should assume greater importance in the interpretation of genotoxicity data. Toxic events associated empirically with the non-genotoxic aspects of chemical carcinogenicity are discussed.


Pathological markers for non-genotoxic agent-associated carcinogenesis.

A variety of positive or negative enzyme altered foci have been proposed as preneoplastic marker lesions in the rat liver. Frozen sections are required in some cases. We have compared the suitability of various histochemically or immunohistochemically demonstrated markers and concluded that immunohistochemically-stained glutathione S-transferase placental form (GST-P) positive foci are particularly useful for practical application in risk assessment. Advantages include ease of quantitative foci analysis on a number of samples since acetone or formalin-fixed paraffin blocks can be used and clear contrast of foci against the surrounding liver tissue facilitates recognition. We have established a liver medium-term bioassay model of 8 weeks' duration using diethylnitrosamine as an initiator and GST-P positive foci as the endpoint lesions. At present, 58 non-genotoxic chemicals for which carcinogenicity data are available have been examined and many carcinogenic agents, mostly liver carcinogens, have been satisfactorily detected as having carcinogenic potential. Exceptional examples are two peroxisome proliferators, clofibrate and DEHP. For these chemical, several peroxisomal enzymes such as catalase and enoyl CoA hydratase have been tested as markers.


Non-genotoxic factors in the carcinogenetic process: problems of detection and hazard evaluation.

In the classical two-stage models of carcinogenesis, initiation has been usually related to a DNA-damage/gene-mutation event, while promotion has been related to the non-genotoxic effects of clonal expansion of preneoplastic cells and/or modulation of cell differentiation. It is now clear that the process of carcinogenesis is linked to more than one irreversible alteration in the genome. Likewise, we can envisage that non-genotoxic events can take place after perhaps 0, 1, 2 or more irreversible alterations in the genome. Initiating and promoting activities of a chemical can be considered clearly separated in theory, but in practice, the chemicals we work with only rarely will be purely of the genotoxic or non-genotoxic type. We will discuss an empirical approach to classify genotoxic or prevalently non-genotoxic chemical carcinogens. For prevalently non-genotoxic carcinogens we will analyze what fraction of them can be detected as promoters of in vivo rat liver carcinogenesis. We will analyze carcinogenic potency of genotoxic and non-genotoxic carcinogens.


Risk assessment of non-genotoxic carcinogens.

Rates of cell proliferation, cell death, and cell differentiation affect the risk of cancer profoundly. An increase in cell proliferation rates leads to an increase in mutation rates per unit of time, which, in turn, leads to an increase in the risk of cancer. An increase in cell division rates relative to death or differentiation rates may lead to an increase in the population of critical target cells, which, again, leads to an increase in cancer risk. These fundamental principles are well illustrated by the rodent liver model for carcinogenesis. In this paper, we shall briefly discuss some of the consequences of incorporating cell proliferation kinetics into quantitative models of cancer risk assessment. Consideration of cell kinetics can shed light on apparently paradoxical observations such as, e.g. the observation that the administration of two different promoters may lead to the same volume fraction in the rodent liver, with one promoter giving rise to a large number of small foci, and the other to a small number of large foci. Some consequences of explicitly considering cell proliferation kinetics in malignant foci are briefly discussed.


Chemicals classified by IARC: an investigation of some of their toxicological characteristics.

Chemicals classified by the IARC to its Groups 1, 2A, 2B and 3 were examined in an attempt to identify characteristics of their behaviour in experimental studies of carcinogenicity, genotoxicity and acute, mammalian toxicity that correlate with those categories. Only those agents for which carcinogenic potency information was available were studied. For both mice and rats, greater proportions of chemicals were potent carcinogens if they had been categorized in Group 1 (human carcinogens) than if they had been put into one of the other categories. Not surprisingly, there was a weak association between carcinogenic potency and acute toxicity. Mice were especially sensitive to tumour induction by halides, while the lower sensitivity of rats to any carcinogenic effect of halides could be due in part to the higher systemic toxicity of halides in this species: a reduced differential of toxic and carcinogenic doses decreases the dose window in which carcinogenic effects may be demonstrated. It was notable that the human carcinogens were active in those genotoxicity tests with higher specificity for identifying rodent carcinogens. Predictive assays for carcinogenicity considered to have high specificity were in vivo cytogenetic, hepatocyte unscheduled DNA synthesis and Salmonella (5 commonly used strains) and mammalian cell hprt locus mutation assays. None of the relationships was strong enough to form the basis of a simple categorization process, but they could serve to alert investigators to chemicals of special toxicological interest and importance.


Problems and opportunities in toxicity testing arising from species differences in xenobiotic metabolism.

The processes of metabolism and disposition have a major bearing upon the biological properties of xenobiotics, determining both the chemical natures and target concentrations of the compound-derived materials in the body. The occurrence of major quantitative and qualitative differences between animal species in the metabolism of xenobiotics is well documented. Interspecies differences in metabolism represent a major complication in toxicity testing, being responsible for important differences both in the nature and magnitude of toxic responses. As such, they may be both help and hindrance and this presentation will illustrate both these possibilities with appropriate examples. In particular, these differences represent probably the single greatest complicating factor in the use of animal toxicity data as an indication of potential human hazard. Although it is considered desirable to identify a species which metabolizes the test compound like man, this ideal is generally not attainable. While metabolic and toxicokinetic data should be used in the selection of animal species, in reality the choices are constrained by other major factors such as availability, background pathological knowledge and regulatory acceptability. On the other hand, species differences in metabolism may present exploitable opportunities for insights into mechanisms of toxicity and with appropriate supporting data may thereby increase confidence in the animal-to-human extrapolation. The application of the new technologies of transgenesis to the creation of animals expressing the genes for human drug metabolizing enzymes offers the promise of increasing the opportunities and minimizing the problems presented by species differences in xenobiotic metabolism.


Sex-specific cytochrome P450 as a cause of sex- and species-related differences in drug toxicity.

Male rats are the most frequently used experimental animals in drug toxicity tests. However, there are clear sex-related differences in toxicity of various drugs and chemicals in rats. These differences, in most cases, are closely connected with the sex-related differences in hepatic drug metabolisms. Recent studies indicate the existence of sex-specific cytochrome P450, such as P450-male (2C11) and P450-female (2C12) and P450(6) beta (3A2) in rat livers, and also show that their expression levels are markedly different between male and female rats. The expressions of sex-specific P450s are regulated by growth hormone, thyroid hormone, sex hormones and other chemicals. On the other hand, there are no or few cytochrome P450s that show the sex-related differences in species other than rats and mice. Although there are orthologous cytochrome P450s in viewpoints of amino acid sequence and substrate specificity in experimental animal species and humans, their expressions are not regulated by hormonal factors in most of the species. These differences may cause clear species differences, if male animals are used, in the toxicity caused by various drugs and chemicals. Thus we can predict the sex-related difference in drug toxicity on the basis of difference in the expression levels of sex-specific cytochrome P450s.


Dose-dependent metabolism and dose setting in chronic studies.

Because of the expense involved in conducting chronic studies, limited numbers of animals and dose groups are used. This has given rise to the practice of including as one of the dose groups the "Maximum Tolerated Dose" (MTD). This dose is operationally defined as the highest dose which can be administered to animals without adversely affecting their survival through effects other than cancer. Since many detoxification systems in animals are capacity-limited, they frequently become saturated in MTD studies. This may lead to difficulties in interpreting the results of MTD studies, particularly when it is necessary to estimate the hazard for human populations whose exposure is typically much lower than the MTD. For this reason, it is important to characterize the dose-dependency of absorption, distribution, metabolism, and elimination (pharmacokinetics) of test substances prior to the initiation of a chronic study. This provides a basis for determining the number and spacing of doses to be used in a chronic study. If the appropriate information is collected it may also be possible to develop a physiologically based pharmacokinetic model which facilitates extrapolation of the toxicity results between different species and routes of administration as well as between high and low doses. For instance, methylene chloride and vinyl chloride are predominantly metabolized by saturable oxidative pathway(s) at low exposure concentrations. In each case, the oxidative pathway saturates at exposures much lower than the MTD. Knowledge of the pharmacokinetic behavior of these substances provided a basis for appropriately interpreting the chronic studies which have been conducted with these materials.


Metabolic studies as a basis for the interpretation of metal toxicity.

The toxicity of metal compounds has traditionally been regarded as a function of dose and potency of the metal itself. In recent years, however, it has become clear that several metals and metalloids undergo transformations in mammalian tissues and that metabolism may have important implications in clinical pharmacology, toxicology, and environmental health. In this paper, data obtained from recent metabolic studies are reviewed as a basis for the interpretation of biological effects and kinetic patterns of metals of major importance as environmental pollutants.


Characterization of the effects of aluminum on luciferase biosensors for the detection of ecotoxicity.

Luciferase-based biosensors are becoming increasingly used for environmental monitoring. A transcriptional fusion of the Vibrio harveyi luxAB genes (encoding bacterial luciferase) to the fliC gene of Escherichia coli was constructed and luminescence shown to be induced (in liquid media) in the presence of 1-10 micrograms/ml aluminum, but not copper, iron or nickel. Moreover, luminescence is markedly increased at pH 5.5, where aluminum is more soluble than at pH 7.0. However, aluminum also stimulated luciferase activity when the luxAB genes were located in the xyl operon. This suggests that aluminum stimulates luciferase enzyme activity in vivo. These results are specific to E. coli, as no such aluminum stimulation was observed in the luminescent bacterium V. harveyi. These results have important implications in the generalized use of these clones for environmental monitoring, where aluminum can be present at elevated concentrations.


Cell injury and death caused by bacterial protein toxins.

Bacterial protein toxins, such as Clostridium difficile toxin A and the Escherichia coli cytotoxic necrotizing factor 1 are known to exert their cytotoxic action via a modification of some cytoskeletal components. The changes in actin organization caused by these toxins appear to be the primary events in the mechanism leading to cell death.


Toxicity in animals and safety in humans: the predictive value of animal studies.

Up to the beginning of the 1980s the surveys on adverse reactions to uro-angiographic, iodinated contrast media administered intravenously failed to recognize any significant correlation between the incidence of such reactions and the type and/or dose of administered compound. Consequently the role of pharmaco-toxicological characteristics of the different iodinated molecules was considered negligible in comparison with the individual risk factors causing for the systemic adverse reactions. More recent surveys have shown that the risk of severe adverse reactions is about six times lower with non-ionic than with ionic compounds. These clinical results confirm the findings of animal studies that showed a two- to three-fold greater safety margin for non-ionic compounds than ionic compounds. In view of these data, the predictive value of pre-clinical safety assessment of iodinated contrast agents is re-examined. The interspecies scaling approach is considered useful owing to the well-known and very simple pharmacokinetic behaviour of these compounds both in humans and in animals.


Qualitative and quantitative experimental models to aid in risk assessment for immunotoxicology.

We have previously reported on the design and content of a screening battery using a "tier" approach for detecting potential immunosuppressive compounds in mice [1]. This battery was composed of various immune function, immunopathology and host resistance tests, the results of which could help establish the potential of chemical and biological agents to cause immunosuppression. The data from these studies, which now encompass over 50 compounds, have been analyzed in an attempt to improve future testing strategies and provide information to aid in the risk assessment process. Specifically, the following two issues will be addressed; what are the likelihood(s) for each of the individual tests and testing configurations to accurately identify immunotoxic compounds? and what are the quantitative and qualitative relationships between the immune tests and host resistance assays?


Epidemiology of adverse reactions to intravascular iodinated contrast media.

Non-ionic contrast media cause fewer acute reactions than ionic media but there is insufficient data on mortality rates. An international reference centre is suggested for histological assessment of fatal reactions. Risk factors, delayed reactions, renal, neural, haemic and other miscellaneous reactions are briefly discussed.


The profile evolution of acute severe poisoning in Spain.

About 5% of the patients admitted to emergency departments with poisonings are seriously ill and need admission to intensive care units (ICUs). This paper presents the result of three multicentric studies carried out in Spain during the last ten years. A study was made of 6 ICUs in 1980, 41 in 1987 and 15 in 1990 with a total of 596 patients. The average age (36) remained stable throughout, but the number of female cases decreased each time. The most frequent cause (79%) was attempted suicide. The products used most frequently were therapeutic drugs (82, 71 and 58%) followed by drugs of abuse, and then agricultural, household and industrial products. We observed a progressive decrease in barbiturates (54, 15 and 2%) and an increase in benzodiazepines and cyclic antidepressants. Therapeutic methods used were mostly gastric lavage (64%), activated charcoal (35%), forced diuresis (43%), extracorporeal therapy (11%) and antidotes. The mortality rate fluctuated between 6.4 and 9.1%, being significantly higher with poisoning by non-therapeutic drugs. In conclusion, the most common poisoning admitted to our ICUs was by psychoactive drugs in suicide attempts. We observed the under-use of activated charcoal and an excessive usage of kidney and extracorporeal methods. The prognosis was worse for poisonings with non-therapeutic drugs.


Rapidly developing cocaine-induced peripheral portal liver damage.

Cocaine administration to phenobarbitone treated DBA/2Ha mice caused scattered peripheral portal cell necrosis within 2 h. At 3 h, when this initial lesion had become more widespread, and plasma membrane integrity had been compromised, 15-fold elevations in plasma levels of liver enzymes were observed. Biochemical analysis of liver microsomes showed no changes in the levels of total cytochrome P-450 during the development of hepatic damage, and there was no correlation between microsomal malondialdehyde (MDA), an indicator of lipid peroxidation, and liver damage. These results indicate that as liver damage occurs only 2 h after cocaine administration, prompt action is warranted when cocaine overdose is suspected.


A dual effect of some 5-HT3 receptor antagonists on cisplatin-induced emesis in the pigeon.

In the present study, the emetic effect of the anticancer drug cisplatin, and protective effects of 5-HT3 receptor antagonists against cisplatin emesis were investigated in the pigeon. The experimental setting involved the i.v. administration of drugs and subsequent observation of the percentage of vomiting animals and number of emetic episodes per vomiting animal over a period of 5 h. In some experiments, the 5-HT and 5-HIAA content in tissues was estimated by the HPLC technique. It was observed that cisplatin (2.5-10 mg/kg) is able to induce dose-dependent emesis in the pigeon. 5-HT3 receptor antagonists (500 micrograms/kg) afford partial protection against cisplatin emesis, although some of them, i.e. indolic derivatives and zacopride, display intrinsic emetic activity at doses of 50-500 micrograms/kg. A serotonergic mechanism appears to be involved in both cisplatin- and 5-HT3 receptor antagonist-induced emesis, since pretreatment with an inhibitor of 5-HT synthesis, para-chlorophenylalanine (300 mg/kg x 3 days), is able to hamper vomiting induced by either cisplatin or 5-HT3 receptor antagonists. It is concluded that the intrinsic emetic effects of 5-HT3 receptor antagonists in the pigeon provide pharmacological evidence of species differences in the properties of 5 HT3 receptors.


Production of lipid hydroperoxides and depletion of reduced glutathione in liver mitochondria after acute ethanol administration to rats.

It has been found that acute ethanol (EtOH) intoxication to rats caused approximately 40% depletion of mitochondrial reduced glutathione (GSH). A GSH reduction of similar extent was also observed after the administration to rats of buthionine sulfoximine (BSO), a specific inhibitor of GSH synthesis. The combined treatment of EtOH plus BSO induced a further mitochondrial GSH decrease up to 70% with respect to control. The presence of lipid hydroperoxides in the mitochondrial membrane was observed whenever an additional oxidative stress was associated to a condition of GSH depletion as in the case of EtOH or EtOH plus BSO. Under these conditions a severe derangement in mitochondrial oxidative functions occurred.


Plasma glutathione level in paracetamol daily abuser patients. Changes in plasma cysteine and thiol groups after reduced glutathione administration.

Since plasma reduced glutathione (GSH) seems to reflect liver GSH content, we have assessed plasma GSH in patients using paracetamol daily. In these patients a significant lower plasma GSH concentration was found with respect to controls. After the i.v. administration of GSH free plasma cysteine was 12 fold higher than in basal condition and all the pattern of plasma thiol groups was modified. This work suggests that the possible protective effect of GSH administration is due to the availability of plasma thiol compounds that enter the cell rather than GSH itself.


Properties of bolesatine, a translational inhibitor from Boletus satanas Lenz. Amino-terminal sequence determination and inhibition of rat mitochondrial protein synthesis.

Bolesatine is a toxic monomeric glycoprotein of Mr 63,000 isolated from the mushroom Boletus satanas Lenz. Its N-terminal amino acid sequence was established: NH2-Thr-Trp-Arg-Ile-Tyr-Leu-Asn-Asn-Gln-Thr-Val-Lys-Leu-Ala-Leu-Leu-Leu- Pro- Asn-Gly.... It inhibits protein synthesis in isolated rat mitochondria. After 90 min of preincubation with bolesatine, the incorporation of [14C]-leucine into mitochondrial proteins was inhibited with an IC50 of 530 nM.


Molecular basis for differences in susceptibility to coal workers' pneumoconiosis.

It is generally accepted that there are differences among workers in susceptibility towards the effects of mineral dusts such as silica, coal dust and asbestos. Basic research continues to find new factors involved in the process of pulmonary fibrosis caused by these minerals. In this paper, two hypotheses implicitly generated by recent findings were tested in two case-control studies among coal miners: generation of tumour necrosis factor (TNF) by blood monocytes of miners with coal workers' pneumoconiosis (CWP) and serum type III procollagen peptide (PIIIP) in CWP. Our data indicate that both parameters can be used as biological markers for early diagnosis of CWP. A follow-up study is described in which the predictive power of increased TNF release and serum PIIIP as risk factor to develop lung fibrosis will be assessed.


Behavioral toxicity of guanidinosuccinic acid in adult and young mice.

Guanidinosuccinic acid (GSA), a guanidino compound found to be greatly increased in uremia, was administered by intraperitoneal (i.p.) injection to adult albino mice and to young mice 7, 14 and 21 days old. Epileptogenic and toxic properties were assessed and GSA brain levels following i.p. injection were determined. In adult mice, GSA induced long-lasting generalized clonic and clonic-tonic convulsions in a dose-dependent manner with a CD50 (and 95% confidence interval) of 363 (287-458) mg/kg (n = 35), and an LD50 of 579 (445-756) mg/kg. The CD50 of GSA corresponded with a brain concentration of 56 nmol/g tissue. Electrocorticographic recording in five adult mice revealed epileptiform discharges (spikes, spike-waves, and polyspike-waves) which appeared concomitant with the convulsions. When young mice were i.p. injected with a (for adults) subconvulsive dose of GSA (250 mg/kg), an age-dependent decrease was noted in GSA-induced convulsions and in the resulting brain concentration. The presented findings suggest that GSA could be an important uremic toxin which could contribute to the epileptic symptomatology in uremia.


Three-generation reproductive studies of a synthetic pyrethroid--cyhalothrin.

The present study was carried out to understand the effect of cyhalothrin, a synthetic pyrethroid, on reproduction in mice. The study was designed as a three-generation reproduction study. Mice were given 2.5 and 5.0 ppm cyhalothrin/kg body weight orally. Daily cumulative body weight of the pregnant mice, number of pups, and cumulative body weight of pups were noted during the study. Compared with the control, body weight gain of cyhalothrin-treated parent mice of the first generation (for both concentrations) was less. As per Dunnet t-test, the weights of treated mice were comparable with control or were statistically nonsignificant. The other two generations did not show significant changes in body weight gain. Cyhalothrin fed orally did not affect the body weight gain of the pups in any of the generations. There was no significant difference in the viability index in any of the generations. Survival of the pups of dams given cyhalothrin was similar to that in the control.


Safety/risk assessment of pesticides: principles, procedures and examples.

The principles and procedures for the assessment of the safety/risk of chemical used by the relevant WHO and EPA expert groups are outlined. The assessment in terms of acceptable daily intakes (ADIs) and reference doses (RfDs) of 25 pesticides is listed. The pesticides assessed are acephate, alachlor, amitrole, azinphos-methyl, benomyl, biphenthrin, bromophos, chlordane, chlorthalonil, cyhalothrin, DDT, EPTC, ethion, folpet, fosetyl-al, glyphosate, isofenphos, methomyl, methyl mercury, paraquat, phosphamidon, systhane, terbutyn, tribultyltin oxide, and vinclozin. In addition, their critical effects, the no-observed-effect levels and the size of the safety/uncertainty factors used are also listed to illustrate the diversity of the toxic effects and the resulting assessments. Furthermore, the enormous amount of data reviewed and the complex scientific judgement involved are also indicated. Considering the various uncertainties existing, the ADIs and RfDs do not differ appreciably in most instances. However, marked differences exist between the ADIs and RfDs of DDT and chlordane. It is suggested that re-evaluation be done on these, and perhaps other, chemicals.


The mechanisms and evaluation of chemically induced allergy.

Many chemicals are able to induce contact allergy, a delayed-type hypersensitivity reaction. Some of these have, in addition, the potential to cause respiratory allergy, an immediate hypersensitivity reaction effected by IgE antibody. The characteristics of immune responses provoked by chemical allergens determine the type of hypersensitivity reaction which will predominate. Recent evidence indicates that contact and respiratory allergens stimulate qualitatively different immune responses in mice consistent with the selective activation of T helper (TH) cell subpopulations. Contact allergens which lack the potential for respiratory sensitization preferentially activate TH1 cells which effect delayed-type hypersensitivity reactions. A soluble product of TH1 cells, interferon gamma (IFN-gamma), inhibits IgE antibody responses, and thereby the induction of respiratory sensitization. Conversely, chemical allergens which are known to cause respiratory hypersensitivity stimulate preferentially TH2-type responses. TH2 cells promote the development of respiratory allergy through the production of interleukin 4 (IL-4), a cytokine which is required for the initiation and maintenance of IgE responses. Although the molecular basis for the selective activation by contact and respiratory allergens of TH1 and TH2 cells, respectively, awaits clarification, these qualitative differences in immune response provide opportunities for the identification and evaluation of chemical sensitizers.


Athymic experimental animals in pharmaco-immunological research.

Immunodeficient animals--the nude mouse and the nude rat--allow studies of drug action and possible side effects without interference from the immune system. Comparative investigations in athymic and euthymic animals allowed us to elucidate the role of T-lymphocytes in the pathogenesis of streptozotocin-induced diabetes mellitus in mice, and the importance of NK-cells as effectors in guanethidine-induced sympathectomy in the rat. It is suggested that immunodeficient animals should be included in toxicological studies of xenobiotics.


Cellular and humoral mechanisms of immunotoxicological tissue manifestations induced by immunotoxic drugs.

The adverse effects on the immune system resulting from acute and subchronic exposure to immunotoxic compounds can be divided into the following three categories. One is the tissue manifestations induced by either immunosuppressive or immunomodulative compounds through the interactions of the drug or its metabolites with cellular/humoral constituents directly or indirectly. For the detection, the functional tests especially NK activity in short-term administration were found to be valuable in the assessment of immunotoxicity of drugs in comparison with hematological and morphological alterations. The second is manifested as an allergic reaction, consisting of three sequential events: (1) sensitization by the exposure of a drug primarily recognized as an antigen; (2) the interaction of antigen and antibody and/or sensitized mononuclear cell (M), and its sequelae, which initiate the chain of events leading to tissue injury: and (3) tissue injury and inflammation. The third is manifested as autoimmunity. Of these, in the first two reactions, a primary manifestation seems to be cellular injury. In this experiment, the morphological and biochemical changes occurring in the cultured cells were analyzed when the compound was added to cultured cells obtained from (a) normal animals, (b) drug-sensitized animal models prepared by using drug-protein conjugates, and (c) animals administered drugs at the dosage equivalent to therapeutic dose. When a test compound was added to short cultured sensitized M, morphological responses observed were characterized by either diffuse swelling of the cells and decrease of pseudopod movement, or gradual shrinking. No such changes were observed in non-sensitized M. In contrast, when immunosuppressants or immunomodulators were used, increases of pseudopod movement and pinocytosis, and excitation of cytoplasmic movement, were observed in both sensitized and non-sensitized M if compounds were cytotoxic. In both cases, inflammatory cytokines responsible for tissue damage were released from the M. Little release of these factors was observed from the non-stimulated cells. For the third category, an attempt is being made to analyze autoimmune disorders immunotoxicologically by using experimentally induced MDP-arthritis in rodents.


Increased detection of hepatitis C virus (HCV)-infected blood donors by a multiple-antigen HCV enzyme immunoassay.

A new, multiple-antigen enzyme immunoassay (EIA-2) for hepatitis C virus (HCV) antibodies was evaluated in parallel with the previously available c100-3 HCV EIA (EIA-1) in 14,068 volunteer blood donors as well as in 25 cases of transfusion-associated hepatitis C for which recipient and donor samples were available. When compared to EIA-1, the EIA-2 was more sensitive in detecting HCV-infected blood donors. The EIA-2 detected an additional 1 in 1000 EIA-1-negative, surrogate marker-negative donors who were infected with HCV as demonstrated by polymerase chain reaction (PCR). The specificity of the EIA-2 was comparable to that of the EIA-1, but the two tests appear to detect different populations of false-positive donors. Recombinant immunoblot assay-indeterminate donors were detected five times more frequently by the EIA-2; PCR demonstrated that 21 percent of these donors were infected with HCV. The greater sensitivity of EIA-2 was also found in 25 transfusion recipients with non-A, non-B hepatitis; however, in 16 percent of these cases of posttransfusion HCV infection, the EIA-2 failed to detect an HCV-seropositive donor. These data indicate that EIA-2 testing will significantly reduce, but probably not eliminate, the risk of transfusion-associated HCV infection; we estimate this residual per-unit risk to be 1 in 2000 to 1 in 6000 units transfused. On a national level, it is projected that the replacement of the anti-HCV EIA-1 with the EIA-2 will initially prevent up to 40 additional cases of transfusion-associated hepatitis C per day.


Promotion of high school blood donations: testing the efficacy of a videotaped intervention.

"Life to Life," an 11-minute videotape based on social learning principles, was used by 10 blood centers in presentations to 4970 high school students one week before school blood drives. At each school, some students saw the videotape and others attended a blood center's customary presentation. Students also completed a brief questionnaire assessing donation attitudes, donation history, and intent to donate. The videotape accounted for a relative increase of 18.7 percent in donations even when other factors were not controlled for. Results were analyzed with logistic models and showed a consistently positive effect over all models used. For students who had never donated, the estimated odds ratio for actual donation (videotape:control) was 1.528. When the model included both type of presentation and ethnicity, the relative increase in donation over that after the blood centers' usual presentation was 69.8 percent for first-time donors. Among previous donors considered alone, the effect on donation was not significant. Whatever their donor history, students who viewed the videotape showed significantly more positive attitudes toward donation and had greater intention to donate than students who saw the blood centers' standard presentations. These results suggest that this videotape is a useful tool for recruitment of high school blood donors.


Frequency of delayed hemolytic transfusion reactions following antibody screening and immediate-spin crossmatching.

In view of the continuing controversy regarding the use of immediate-spin crossmatch procedures in preparing blood for transfusion to patients in whom unexpected clinically significant antibodies have not been found by antibody screening by the indirect antiglobulin test (IAT), a review of 8 years' experience with such a policy was conducted. In that period, 54,725 units of packed red cells or whole blood were transfused to 10,146 patients. Four clinically overt delayed hemolytic transfusion reactions and 18 clinically silent delayed serologic transfusion reactions were found. In 3 of the 22 patients, the offending antibody(ies) were detectable in the pretransfusion serum by an enzyme IAT, but none was detectable by routine saline IAT against either a three-cell screening panel or the transfused cells. Thus, the incorporation of saline indirect antiglobulin crossmatch would not have prevented the delayed reactions. It can be concluded that the use of a saline indirect antiglobulin crossmatch offers no significant advantage over the current policy of using only immediate-spin crossmatch for those patients whose pretransfusion serum gives negative results in a three-cell screen using a saline IAT.


Immunohematologic complications of ABO-unmatched liver transplants.

Transplantation of ABO-unmatched livers has been associated with the development of donor-derived antibody (DDAb) and hemolysis. Nine (22%) of 41 consecutive patients undergoing liver transplantation at our institution received 10 ABO-unmatched livers. Five (56%) of nine patients developed DDAbs and hemolysis. All five patients were group A1 and received group O livers. DDAbs appeared a mean of 9.2 +/- 2.8 (1 SD) days after surgery and persisted for 15.2 +/- 10.3 days. All patients with DDAbs developed hemolysis. During the period when DDAbs were demonstrable, the hemoglobin dropped by a mean of 4.8 g per dL (48 g/L), and the patients were transfused with a mean of 7.8 +/- 2.3 units of group O red cells. One patient with hemolysis underwent exchange transfusion for acute renal failure. Patients with hemolysis required significantly more red cells postoperatively (15.0 vs. 6.9 units, p = 0.04) than did ABO-matched patients. None of the parameters examined (age, recipient or donor gender, secretor status, rejection, or donor isoagglutinin titers) were predictive of DDAb or hemolysis, although hemolysis occurred in three of four cases in which donor serum IgG anti-A titers were > or = 128, as opposed to one of four cases in which titers were < 128. Because recipients of ABO-unmatched livers are at high risk for transiently developing DDAb and hemolysis with associated morbidity, the prophylactic use of donor-type red cells for surgery and after operation is justified.


HLA antibody enhancement by double addition of serum: use in platelet donor selection.

The identification of HLA class I alloantibodies is important for organ transplantation and platelet transfusion in alloimmunized patients. Because microcytotoxicity testing against frozen trays of lymphocyte panels is rapid and efficient for determining specificities of unknown antibodies, a simple method was devised to increase test sensitivity to weak antibodies. Standard anti-human globulin (AHG)-facilitated microcytotoxicity was modified by the insertion of a double addition-of-serum (DAS) step, and reagent and patient's sera were evaluated by both methods. DAS modification increased antibody titers and, more significantly, made the identification of weak specificities easier because of the twofold to threefold increase in reactivity rates (29-42% for AHG vs. 75-82% for DAS) of panel cells that were expected to be positive, while low (approx. 1%) "extra" reaction rates were maintained for cells that were expected to be negative. DAS was relatively unaffected by variations in serum volumes or target cell preparation, and its use did not significantly increase test time or costs. In a program of platelet donor selection driven by donor antibody rather than donor-recipient antigen matching, DAS greatly facilitated platelet transfusion support for alloimmunized patients.


Enter a new post-translational modification: D-amino acids in gene-encoded peptides.

The post-translational processing of peptides plays a key role in conferring biological activity on those peptides. Recently, ribosomally made peptides that contain D-amino acids at specific positions have been discovered in microorganisms as well as in vertebrates and invertebrates. This points to yet another strategy of circumventing stereochemical limitations imposed by the genetic code and conveying biological activity to otherwise inert molecules.


Expression of the Tn antigen in myelodysplasia, lymphoma, and leukemia.

Expression of the normally cryptic blood group antigen Tn has occasionally been reported in hematologic disease, but the true frequency of this change is not known. A mouse monoclonal antibody (FBT3) and immunohistochemistry were used to examine expression of the Tn antigen. Expression was not detected in 35 normal bone marrow aspirates examined, but it was detected in 5 of 725 abnormal bone marrow aspirates, including 2 (3.6%) of 55 cases of de novo acute nonlymphocytic leukemia and 2 cases that terminated in acute nonlymphocytic leukemia. In two patients, one with acute myeloblastic leukemia and the other in blast transformation of chronic myeloid leukemia, the Tn antigen was expressed on 2 percent of blast cells. In one case of non-Hodgkin's lymphoma, 4 percent of normal myeloid cells expressed the antigen. In the other two cases, one of acute myelomonocytic leukemia and the other of myelodysplasia, only 2 to 8 percent of myeloid and erythroid cells initially were Tn positive. Subsequent serial immunohistochemical studies of bone marrow aspirates and peripheral blood in these two cases showed increasing numbers of Tn-positive erythroid and myeloid cells 8 to 12 months before polyagglutination was detected serologically. Tn-positive cells increased to > 90 percent in the terminal phase in both cases of both diseases. The results suggest that Tn expression in these two patients may have conferred a growth advantage to the cells and could be related to disease progression.


A serologic relationship among the NFLD, BOW, and Wu red cell antigens.

A survey of sera containing antibodies to multiple low-incidence antigens revealed a variety of patterns of reactions with NFLD+, BOW+, and Wu+ red cell samples. Although NFLD, BOW, and Wu are distinct antigenic determinants (International Society of Blood Transfusion numbers 700.37, 700.46, and 700.13, respectively), their ability to absorb and elute "crossreactive" antibodies indicates a serologic and possibly a genetic relationship among the three.


Did the ancestral globin gene of plants and animals contain only two introns?

All vertebrate globin genes contain two introns, while plant globin genes contain three. It is widely thought that the plant gene structure reflects the structure of the primordial globin gene and that a common ancestor of all animals lost the central intron shortly after the divergence of plants and animals more than one billion years ago. The recent discovery of a discordant central intron in some animal globin genes suggests that this model is incorrect. We propose that the typical vertebrate two-intron gene structure is the primordial eukaryotic form, and that following the divergence of plants and animals, a common ancestor of plants gained a central intron in the globin gene.