Catherine S. Sprankle

Variation in expression of genes used for normalization of Northern blots after induction of cell proliferation

Quantitative knowledge of gene expression can provide valuable information for understanding the action of chemicals that alter cell proliferation and cancer. Accurate quantification of mRNA levels requires the normalization of the gene of interest to a gene with transcriptional levels that do not vary through the cell cycle or with a particular treatment. Changes in expression were examined in proliferating or non‐proliferating rat liver for three constitutively expressed ‘housekeeping’ genes commonly used to normalize mRNA levels from Northern blots. In addition, a direct method of quantifying poly(A)+ mRNA by hybridization with a radiolabelled polythymidylate—poly(T)—probe was compared with traditional methods. Hepatocyte cytolethality and a subsequent wave of hepatocyte proliferation were induced in male Fischer‐344 rats by treatment with a single gavage dose of carbon tetrachloride. Induced cell proliferation peaked at 48 h after treatment. Expression of the housekeeping genes actin, glyceraldehyde‐3‐phosphate‐dehydrogenase (GAPDH) and albumin, as well as the proto‐oncogene H‐ras, was determined by Northern blot analysis at times from 0.5 h to 4 days after treatment. Time‐dependent changes were observed in the expression of these genes relative to the levels observed in the untreated control animals. Actin expression peaked at 3.4‐fold over control and GAPDH expression was increased by 1.9‐fold over control. Albumin mRNA levels varied the least, 1.4‐fold over control, indicating that this gene is more appropriate than actin or GAPDH for normalization of proto‐oncogene expression under experimental conditions that induce cell proliferation in rat liver. The direct quantification of poly(A)+ mRNA using a poly(T) probe was not influenced by the induction of cell proliferation. This method may be useful when the expression of housekeeping genes is affected by treatment.

Expression of myc, fos and Ha-ras associated with chemically induced cell proliferation in the rat liver

Abstract. Events secondary to induced cell proliferation may play a role in the carcinogenic process. These studies investigated the expression of genes associated with growth control in response to two types of cell proliferation stimuli in the livers of male F344 rats. Regenerative hepatocyte proliferation after partial hepatectomy or a single dose of carbon tetrachloride, and mitogenic liver hyperplasia induced by a single dose of pheno‐barbital or WY‐14,643 were assessed by thymidine incorporation and quantitative autoradiography. The expression of myc, fos, and Ha‐ras was evaluated by Northern blot analysis of liver derived poly(A)+ mRNA from these same animals. After each treatment, the level of hepatocyte proliferation (labelling index 4–32%) was observed to peak between 24 and 48 h and return to control values by 8 days. In every case, a peak in myc expression was seen between 0.5 and 18 h depending on the proliferative stimulus treatment. A large peak infos expression was seen at 0.5–2 h but only with the cytotoxic and regenerative proliferative treatments partial hepatectomy or carbon tetrachloride. A broad peak in Ha‐ras expression was observed 12 to 36 h after each treatment. These data demonstrate transient expression of these genes following the synchronous induction of hepatocyte proliferation. The increased expression offos upon treatment with cytotoxicants, but not mitogens, suggests different modes of growth regulation that may be important in understanding the induction of cell proliferation by these two types of agents.

Long-term mutagenicity studies with chloroform and dimethylnitrosamine in female lacI transgenic B6C3F1 mice

The weight of evidence indicates that chloroform induces cancer in the female B6C3F1 mouse liver via a nongenotoxic‐cytotoxic mode of action. However, it is probable that DNA damage occurs secondary to events associated with cytolethality and regenerative cell proliferation. The purpose of the present study was to evaluate the potential mutagenic activity of chloroform in the B6C3F1 lacI transgenic mouse liver mutagenesis assay including mutagenic events that might occur secondary to cytolethality. The positive control, dimethylnitrosamine (DMN) is a DNA‐reactive mutagen and carcinogen. DMN‐induced mutations were anticipated to require only a brief exposure and without further treatment were predicted to remain unchanged over time at those frequencies. Chloroform‐induced mutations secondary to toxicity were anticipated to require longer exposure periods and to occur only under conditions that produced sustained cytolethality and regenerative cell proliferation. Female B6C3F1 lacI transgenic mice were treated with daily doses of 2, 4, or 8 mg/kg of DMN by gavage for 4 days and then held until analysis 10, 30, 90, and 180 days postexposure. Livers from DMN‐treated mice exhibited a dose‐related 2‐ to 5‐fold increase over control mutant frequencies and remained at those levels for 10 through 180 days postexposure. Thus, following the initial induction by DMN no selective mutation amplification or loss was seen for this extended period of time. Female B6C3F1 lacI mice were exposed daily for 6 hr/day 7 days/week to 0, 10, 30, or 90 ppm chloroform by inhalation, representing nonhepatotoxic, borderline, or overtly hepatotoxic chloroform exposures. Timepoints for determination of lacI mutant frequency were 10, 30, 90, and 180 days of exposure. No increase in lacI mutant frequency in the liver was observed at any dose or timepoint with chloroform, indicating a lack of DNA reactivity. DNA alterations secondaryto toxicity either did not occur or were of a typenot detectable by lacI mutant frequency analysis,such as large deletions. Environ. Mol. Mutagen. 31:248–256, 1998

Chloroform-induced cytotoxicity and regenerative cell proliferation in the kidneys and liver of BDF1 mice

In a 2-year chloroform inhalation bioassay, an increased incidence of tumors was observed in the kidneys of male BDF1 mice and the liver of female BDF1 mice exposed to the highest exposure concentration of 90 ppm. To investigate the role of cytotoxicity and regenerative cell proliferation in tumor formation, male and female BDF1 mice were exposed to chloroform vapor concentrations of 0, 0.3, 5, 30, or 90 ppm 6 h/day for 4 days. Bromodeoxyuridine (BrdU) was administered via osmotic pumps implanted 3.5 days prior to necropsy, and the labeling index (LI), or percentage of cells in S-phase, was quantified using BrdU immunohistochemistry. To assess longer-term responses, additional male mice were exposed 5 days/week for 2 weeks to 0, 30, or 90 ppm. Degenerative lesions and an increase in the LI of seven- to ten-fold over controls were observed in the kidneys of male but not female mice exposed to 30 or 90 ppm. Liver lesions and increased hepatocyte LI were observed in male mice exposed to 30 or 90 ppm and in female mice exposed to 90 ppm. In the 2-week exposure groups 40% of the 30 ppm group and 80% of the 90 ppm group died with severe kidney damage, indicating that both 30 and 90 ppm exceed a maximum tolerated dose. Thus, in the 2-year bioassay chloroform concentrations had to be stepped-up over a period of weeks in order for the male mice exposed to 30 or 90 ppm to survive. The extrapolation of tumor data from such an unusual procedure is questionable. These observations are consistent with a substantial database that indicates that tumor induction by chloroform occurs via a non-genotoxic-cytotoxic mode of action and is secondary to organ-specific toxicity. These data further support the premise that doses that do not induce regenerative cell proliferation do not present an increased risk of cancer.

In vitro to in vivo extrapolation for high throughput prioritization and decision making

In vitro chemical safety testing methods offer the potential for efficient and economical tools to provide relevant assessments of human health risk. To realize this potential, methods are needed to relate in vitro effects to in vivo responses, i.e., in vitro to in vivo extrapolation (IVIVE). Currently available IVIVE approaches need to be refined before they can be utilized for regulatory decision-making. To explore the capabilities and limitations of IVIVE within this context, the U.S. Environmental Protection Agency Office of Research and Development and the National Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods co-organized a workshop and webinar series. Here, we integrate content from the webinars and workshop to discuss activities and resources that would promote inclusion of IVIVE in regulatory decision-making. We discuss properties of models that successfully generate predictions of in vivo doses from effective in vitro concentration, including the experimental systems that provide input parameters for these models, areas of success, and areas for improvement to reduce model uncertainty. Finally, we provide case studies on the uses of IVIVE in safety assessments, which highlight the respective differences, information requirements, and outcomes across various approaches when applied for decision-making.

Alternative approaches for identifying acute systemic toxicity: Moving from research to regulatory testing

Acute systemic toxicity testing provides the basis for hazard labeling and risk management of chemicals. A number of international efforts have been directed at identifying non-animal alternatives for in vivo acute systemic toxicity tests. A September 2015 workshop, Alternative Approaches for Identifying Acute Systemic Toxicity: Moving from Research to Regulatory Testing, reviewed the state-of-the-science of non-animal alternatives for this testing and explored ways to facilitate implementation of alternatives. Workshop attendees included representatives from international regulatory agencies, academia, nongovernmental organizations, and industry. Resources identified as necessary for meaningful progress in implementing alternatives included compiling and making available high-quality reference data, training on use and interpretation of in vitro and in silico approaches, and global harmonization of testing requirements. Attendees particularly noted the need to characterize variability in reference data to evaluate new approaches. They also noted the importance of understanding the mechanisms of acute toxicity, which could be facilitated by the development of adverse outcome pathways. Workshop breakout groups explored different approaches to reducing or replacing animal use for acute toxicity testing, with each group crafting a roadmap and strategy to accomplish near-term progress. The workshop steering committee has organized efforts to implement the recommendations of the workshop participants.

Differential display identified changes in mRNA levels in regenerating livers from chloroform-treated mice.

The nongenotoxic‐cytotoxic carcinogen chloroform induces liver necrosis, regenerative cell proliferation, and, eventually, liver tumors in female B6C3F1 mice when administered by gavage at doses of 238 or 477 mg/kg/d. Administration of 1800 ppm of chloroform in the drinking water results in similar daily doses but does not produce liver toxicity or cancer. The differential‐display technique was used to compare the expression of a subset of mRNAs in normal (control) and regenerating liver after chloroform‐induced toxicity to define the proportion of genes whose expression changes under hepatotoxic conditions and to identify the genes that might play a role in regeneration and perhaps cancer. RNA was purified from the livers of female B6C3F1 mice after 4 d or 3 wk of gavage treatment with 3, 238, or 477 mg/kg/d of chloroform or treatment with 1800 ppm chloroform in drinking water. There was a remarkably high degree of consistency of gene expression among the animals and across dose and treatment groups as visualized by the differential‐display technique. Of the 387 bands observed, only four (about 1%) changed expression in regenerating liver. The genes were assigned locus names by GenBank after sequence submission. The genes with increased mRNA levels as confirmed by northern blot analysis were MUSTIS21, a mouse primary response gene induced by growth factors and tumor promoters; MUSMRNAH, a gene highly homologous to a human gene isolated from a prostate carcinoma cell line; and MUSFRA, a novel gene. The novel gene MUSFRB exhibited decreased mRNA levels. No change in expression was seen among control mice given the nontoxic regimens of 3 mg/kg/d chloroform or 1800 ppm chloroform in drinking water, indicating that changes in expression were associated with toxicity and regeneration rather than chloroform per se. These genes and others that may be identified by expanding this approach may play a role in regeneration and perhaps in the process of chloroform‐induced carcinogenesis in rodent liver. Mol. Carcinog. 20:288‐297, 1997.

Levels of myc, fos, Ha-ras, met and hepatocyte growth factor mRNA during regenerative cell proliferation in female mouse liver and male rat kidney after a cytotoxic dose of chloroform

Chloroform is a liver carcinogen in mice and a kidney carcinogen in rats. It is thought to act through a non-genotoxic-cytotoxic mode of action. Changes in expression of growth control genes accompanying chloroform-induced cytolethality and regeneration may play a part in the development of chloroform-induced tumors. In this experiment, we examined the levels of the myc, fos, Ha-ras, met and hepatocyte growth factor mRNA in livers of female B6C3F(1) mice and kidneys of male F-344 rats to detect changes in gene expression following a single, cytotoxic gavage dose of chloroform in corn oil. Poly A+ RNA was purified from homogenates of livers of mice treated with 350mg/kg chloroform and kidneys of rats treated with 180 mg/kg chloroform and used for Northern blot analysis. Livers of female mice showed large transient increases in levels of myc and fos mRNA while levels of Ha-ras, met and the hepatocyte growth factor gene mRNA remained near control levels. In the male rat kidney, levels of myc mRNA increased after treatement, while levels of mRNA of all other genes examined remained near control levels. This pattern of gene expression is consistent with that induced by other cytotoxic carcinogens and suggest that alteration of the myc and fos genes could be involved in the regenerative cell proliferation that ultimately could play a role in chloroform-induced tumors.

An Integrated Chemical Environment to Support 21st-Century Toxicology

Summary: Access to high-quality reference data is essential for the development, validation, and implementation of in vitro and in silico approaches that reduce and replace the use of animals in toxicity testing. Currently, these data must often be pooled from a variety of disparate sources to efficiently link a set of assay responses and model predictions to an outcome or hazard classification. To provide a central access point for these purposes, the National Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods developed the Integrated Chemical Environment (ICE) web resource. The ICE data integrator allows users to retrieve and combine data sets and to develop hypotheses through data exploration. Open-source computational workflows and models will be available for download and application to local data. ICE currently includes curated in vivo test data, reference chemical information, in vitro assay data (including Tox21TM/ToxCast™ high-throughput screening data), and in silico model predictions. Users can query these data collections focusing on end points of interest such as acute systemic toxicity, endocrine disruption, skin sensitization, and many others. ICE is publicly accessible at https://ice.ntp.niehs.nih.gov. https://doi.org/10.1289/EHP1759

Expression of the hepatocyte growth factor and c-MET genes during furan-induced regenerative cell proliferation in the livers of B6C3F1 mice and F-344 rats

Abstract. Chronic administration of furan has been reported to produce hepatocellular carcinomas in male and female mice and male rats and cholangiocarcinomas in male and female rats. The weight of evidence indicates that furan is not genotoxic and tumours appear to arise secondary to initiation and promotional events associated with furan‐induced necrosis and regenerative cell proliferation. Identification of altered patterns of gene expression related to these events may provide insight into some of the underlying factors involved in this carcinogenic process. Hepatocyte growth factor (HGF) and its receptor, the protein product of the c‐met oncogene, play an important role in regenerative growth of liver. These experiments examined the relationship between induced cell proliferation and expression of the HGF and c‐met genes in liver after treatment with furan. Male rats were administered a single necrosis‐inducing dose of furan, and male mice and male and female rats were treated daily with furan by gavage for up to six weeks under conditions of the cancer bioassay. Northern blot analysis of mRNA isolated from livers of those rats administered a single dose of furan detected a transient increase in expression in the HGF gene associated with the resulting wave of regenerative cell proliferation, but no increase in the expression of c‐met. No increase in expression of either gene was detected in liver tissue from any of the animals treated for up to six weeks, even though elevated rates of regenerative hepatocyte proliferation were sustained. Thus, given the detection limits of the techniques used here, sustained increases in expression of these genes are not required to maintain the regenerative response.