Development in in vitro toxicology

There are three principal pressures driving the development of in vitro toxicology: (1) the need for more efficient testing systems to cope with the large number of xenobiotics currently being developed; (2) public pressure to reduce animal experimentation; and (3) a need for a better understanding of the mechanisms of toxicity. Within this, in vitro toxicology is focused on local, systemic, and target-organ toxicity. It is becoming increasingly apparent that a step or decision-tree approach using input of a variety of experimental data (physicochemical properties, biokinetics, cytotoxicity) provides the most efficient system for predicting toxicity. Examples of the use of in vitro toxicity systems for prediction of systemic toxicity and target-organ (liver) toxicity are presented.Originally presented at ECCP 93.     

Preantral follicle culture as a novel in vitro assay in reproductive toxicology testing in mammalian oocytes

The most common genetic disorder in humans, trisomy, is caused predominantly by errors in chromosome segregation during oogenesis. Isolated mouse oocytes resuming meiosis and progressing to metaphase II in vitro have recently been used to assess targets, aneugenic potential and sensitivity of oocytes to chemical exposures. In order to extend in vitro maturation tests to earlier stages of oogenesis, an in vitro assay with mouse preantral follicle cultures has been established. It permits the identification of direct and also indirect effects of environmental chemicals on the somatic compartment, the follicle and theca cells, that may lead to disturbances of oocyte growth, maturation and chromosome segregation. Early preantral follicles from prepubertal female mice are cultured in microdroplets for 12 days under strictly controlled conditions. The follicle-enclosed oocytes resume maturation, develop to metaphase II and become in vitro ovulated within 16 h after a physiological ovulatory stimulus with recombinant human gonadotrophins and epidermal growth factor. These oocytes grown and matured in vitro possess normal barrel-shaped spindles with well-aligned chromosomes. Their chromosomes segregate with high fidelity during anaphase I. The model aneugen colchicine induced a meiotic arrest and aneuploidy in these in vitro grown, follicle-enclosed oocytes in a dose-dependent manner, comparable to in vivo tests. Therefore, preantral follicle culture appears to provide an effective and reliable method to assess the influences of environmental mutagens, pharmaceutical agents and potentially endocrine disrupting chemicals on the fidelity of female meiosis.     

Homogeneous populations of Artemia nauplii and their potential use for in vitro testing in developmental toxicology

Efforts have begun to establish test subjects other than the intact pregnant mammal to serve as models for rapid teratology screens. Artemia nauplii transcending instar I to later instars were examined to determine their potential for indicating chemicals as potential developmental hazards and thus prioritizing them for more extensive in vivo testing. Several criteria selected for assessing the system's potential for screening were the ability to: collect homogeneous populations of instar I nauplii; characterize intermediate development by technically simple measurements; and demonstrate development-related differentials in naupliar vulnerability. Homogeneous populations of nauplii were harvested from a flow-through hatching and cold storage system. Nauplii accumulated in the system are stored at 4 degrees C in a quiescent state with little physical (body length, body water volume) and biochemical (DNA and protein levels) change and thus are maintained at instar I. Intermediate development of nauplii transcending instar I to IV was characterized after the onset of 25 degrees C incubation by measuring changes in drinking activity, body length, body water volume, and DNA and protein levels. During the first day of incubation, development was greatest between 6 and 24 hours of incubation. Development-related differentials in naupliar vulnerability were shown by comparing median lethal concentrations (LC50) estimated for cadmium sulfate (CdSO4), mercuric chloride (HgCl2), and sodium azide (NaN3) at various times during incubation. With cadmium and mercury, LC50s decreased as nauplii aged and developed; whereas, with azide, LC50s did not vary. Developing nauplii were differentially vulnerable to cadmium and inorganic mercury. Artemia nauplii transcending instar I to IV appear useful for indicating chemicals that preferentially interact with developmental events.     

The minipig in toxicology

The use of pigs (Sus scrofa) in biomedical research is well established in particular in surgical and physiological research. For years both pigs and minipigs have been used in pharmacology and toxicology to answer specific questions when the more conventional species have been found unsuitable. The development of minipigs has resulted in strains of more manageable size than the domestic pig. Because of their well-accepted physiological and other similarities to humans, minipigs are becoming increasingly attractive toxicological and pharmacological models. There are several strains of minipigs (Göttingen, Yucatan, Sinclair, Hanford and other). This presentation is based on experience primarily with the Göttingen minipigs. In toxicology in Europe minipigs have become very popular for pharmaceutical studies in place of dogs and primates. Minipigs have been shown to be sensitive to a wide variety of drugs and chemicals. It has become obvious that minipigs can be used for all routes of administration, and in many cases are preferable to dogs or primates for metabolic or pharmacological reasons. There are advantages over the traditional non-rodent species in relation to specific responses to particular drug classes. Their use in general toxicology testing employing the continuous intravenous infusion, dermal or inhalation route has been described in detail in the literature. Background data on toxicological endpoints (ophthalmology, clinical pathology, ECG, organ weight, histopathology and reproduction parameters) have been well-established allowing studies to be interpreted. In the context of this conference, histopathology and toxicopathology data of spontaneous or drug-induced origin are available in the scientific literature. Now there is good supply of high-quality minipigs of known disease status. There are advantages over the traditional non-rodent species in relation to the ethical difficulties of use of animals in biomedical research. Consequently, there are scientific, economic and sociological reasons that make minipigs good toxicological and pharmacological models. The principal disadvantage is that toxicity testing in minipigs may require more test compound than the traditional species.     

Genetic toxicology

Problems and achievements in genotoxicology are briefly discussed with special reference to genotoxicologic assays, genotoxicologic risks, modification of genotoxicologic effects, and individual sensitivity to genotoxic agents. The basic terms and concepts of genetic toxicology as well as the main lines of its further development are considered.     

Endocrine toxicology: A Review of the application of endocrinology in experimental toxicology

In the past endocrine toxicology has not been a common subject in routine toxicity studies. However, since the endocrine system is an important integrating system of the body, controlling the major physiological functions, it is important to investigate the mechanism of action of exogenous compounds in endocrine target organs or hormonal target cells. The following procedure is suggested to detect effects on the endocrine system in routine toxicity experiments: (1) determination of the weight of endocrine organs and histology as screening parameters; (2) determination of circulating hormones in combination with morphological or immunocytochemical methods: (3) specific function tests and in vitro methods to determine dysfunction of specific endocrine organs or cells. That the use of such an approach has provided insight into the mechanism of action of chemical compounds will be demonstrated by results of endocrine toxicity studies with the antibiotic compound sulphadimidine, interfering with thyroid hormone synthesis as a secondary mechanism leading to thyroid tumour formation, the androgenic compound trenbolone acetate, used for growth promotion, for which the disturbance of the gonadal function formed the basis for the establishment of the no-observed-hormonal-effect level, the antibacterial compound furazolidone, suspected of having an oestrogenic activity which was hypothesized as the underlying mechanism for the observed mammary tumour formation, and the antimicrobial agent carbadox, used as feed additive for pigs, for which the interference with adrenal function, resulted in a severe disturbance of the water and salt balance in target animals.Originally presented at ECCP 93.     

Endocrine toxicology: A Review of the application of endocrinology in experimental toxicology

In the past endocrine toxicology has not been a common subject in routine toxicity studies. However, since the endocrine system is an important integrating system of the body, controlling the major physiological functions, it is important to investigate the mechanism of action of exogenous compounds in endocrine target organs or hormonal target cells. The following procedure is suggested to detect effects on the endocrine system in routine toxicity experiments: (1) determination of the weight of endocrine organs and histology as screening parameters; (2) determination of circulating hormones in combination with morphological or immunocytochemical methods: (3) specific function tests and in vitro methods to determine dysfunction of specific endocrine organs or cells. That the use of such an approach has provided insight into the mechanism of action of chemical compounds will be demonstrated by results of endocrine toxicity studies with the antibiotic compound sulphadimidine, interfering with thyroid hormone synthesis as a secondary mechanism leading to thyroid tumour formation, the androgenic compound trenbolone acetate, used for growth promotion, for which the disturbance of the gonadal function formed the basis for the establishment of the no-observed-hormonal-effect level, the antibacterial compound furazolidone, suspected of having an oestrogenic activity which was hypothesized as the underlying mechanism for the observed mammary tumour formation, and the antimicrobial agent carbadox, used as feed additive for pigs, for which the interference with adrenal function, resulted in a severe disturbance of the water and salt balance in target animals. Originally presented at ECCP 93.     

Emerging approaches in predictive toxicology

Predictive toxicology plays an important role in the assessment of toxicity of chemicals and the drug development process. While there are several well‐established in vitro and in vivo assays that are suitable for predictive toxicology, recent advances in high‐throughput analytical technologies and model systems are expected to have a major impact on the field of predictive toxicology. This commentary provides an overview of the state of the current science and a brief discussion on future perspectives for the field of predictive toxicology for human toxicity. Computational models for predictive toxicology, needs for further refinement and obstacles to expand computational models to include additional classes of chemical compounds are highlighted. Functional and comparative genomics approaches in predictive toxicology are discussed with an emphasis on successful utilization of recently developed model systems for high‐throughput analysis. The advantages of three‐dimensional model systems and stem cells and their use in predictive toxicology testing are also described. Environ. Mol. Mutagen. 55:679–688, 2014. © 2014 Wiley Periodicals, Inc.     

Oxymetholone: I. Evaluation in a comprehensive battery of genetic toxicology and in vitro transformation assays.

Oxymetholone is generally assumed to be a nongenotoxic carcinogen. This assumption is based primarily on the results of an Ames test, existing data in repeat-dose toxicology studies, and the predicted results of a 2-yr National Toxicology Program (NTP) rat carcinogenicity bioassay. To provide a comprehensive assessment of its genotoxicity in a standard battery of mutagenicity assays, oxymetholone was tested in microbial and mammalian cell gene mutation assays, in an in vitro cytogenetics assay (human lymphocytes), and in an in vivo micronucleus assay. Oxymetholone was also tested in an in vitro morphologic transformation model using Syrian hamster embryo (SHE) cells. These studies were initiated and completed prior to the disclosure of the results of the NTP bioassay. Oxymetholone was tested at doses up to 5,000 microg/plate in the bacterial plate incorporation assay using 4 Salmonella strains and the WP2 uvrA (pKM101) strain of Escherichia coil. There was no induction of revertants up to the highest dose levels, which were insoluble as well as toxic. In the L5178Y tk+/- mouse lymphoma assay, doses up to 30 microg/ml reduced relative survival to approximately 30% with no increase in mutants. Male or female human lymphocytes were exposed in vitro to oxymetholone for 24 hr without S9 or 3 hr with S9 and evaluated for the induction of chromosomal aberrations. There was no increase in aberration frequency over control levels and no difference between male and female cells. Peripheral blood from Tg.AC transgenic mice treated dermally for 20 wk with 0, 1.2, 6.0, or 12.0 mg/day of oxymetholone and from p53 transgenic mice treated orally by gavage for 26 wk with 125, 625, or 1,250 mg/kg/day of oxymetholone was evaluated for micronuclei in polychromatic and normochromatic erythrocytes. There was no difference in micronuclei frequency between control and treated animals. These results confirm that oxymetholone is not genotoxic in a comprehensive battery of mutagenicity assays. In the SHE assay, oxymetholone produced a significant increase in morphologically transformed colonies at dose levels of 13-18 microg/ml. The lack of genotoxicity of oxymetholone, the positive response in the in vitro transformation assay, and the results of transgenic mouse carcinogenicity assays will provide an interesting perspective on the results of an on-going NTP rat carcinogenicity bioassay.     

Inorganic lead toxicology

Lead is a health hazard for all humans. Especially children under the age of six are most at risk for lead poisoning. Lead toxicity causes hematological, gastrointestinal, and neurological dysfunction. Symptoms are usually noted with blood lead greater than 2 micromoles/L. Severe or prolonged exposure may also cause chronic nephropathy, hypertension, and reproductive impairment. Lead inhibits some enzymes, alters cellular calcium metabolism, stimulates synthesis of binding proteins in kidney, brain, and bone, and slows down nerve conduction. Acute lead poisoning is relatively infrequent and results from ingestion of acid soluble lead compounds or inhalation of lead vapors but chronic exposure to low levels of the metal is still a public health issue, especially among some minorities and socioeconomically disadvantaged groups. Lead has been used since prehistoric times, and has become widely distributed and mobilized in the environment. Exposure to and uptake of this non-essential element have consequently increased. Both occupational and environmental exposures to lead remain a serious problem in many developing and industrializing countries and a public health problem of global dimensions.     

Summary of in vitro genetic toxicology assay results: Expected and unexpected effects of recent study design modifications

Key modifications to in vitro genetic toxicology testing have been made in the last 5 years including the use of optimization approaches such as structure–activity relationships and screening assays to identify and eliminate potentially genotoxic chemicals from further consideration, better guidance on cytotoxicity assessment and dose selection, and greater use of p53‐competent human cells. To determine the effect of these changes on testing outcomes, the pattern of positive results across assays conducted by BioReliance from 2005 to 2010 was examined. Data were tabulated for good laboratory practice (GLP)‐compliant Ames, mouse lymphoma (MLA), chromosome aberration in Chinese hamster ovary (CHO) cells, and in human peripheral blood lymphocytes (HPBL) assays along with non‐GLP screening Ames assays. A decrease in percentage of positive results in MLA and CHO chromosome aberration assays was observed, whereas the percentage of positive Ames assays remained consistent. This was not unexpected because MLA and CHO cytogenetic assays have undergone the most substantive changes (e.g., the establishment of the Global Evaluation Factor for the MLA and the use of the relative increase in cell counts in CHO chromosome aberration assays). Over the last 5 years, there has been an increase in the percentage of positive results observed in the chromosome aberration assay in HPBL. It is speculated that this may have led to an increase in HPBL‐positive results if the chemicals routed to HPBL had previous positive genotoxicity results. Another factor may be the lack of a reliable cytotoxicity measurement in the HPBL assay. Environ. Mol. Mutagen., 2012. © 2012 Wiley Periodicals, Inc.