Drug-induced pancreatic injury (DIPI) has become linked in recent years to many commonly prescribed medications from several pharmacological classes. Diagnosis is currently most often focused on identification of acute pancreatitis and generally based on subjective clinical assessment and serum amylase and lipase enzymatic activity, which have been criticized as being insufficiently sensitive and specific. The lack of novel noninvasive biomarkers of DIPI can impede the advancement of drug candidates through nonclinical development and translation into clinical settings. Pancreas-specific microRNAs (miRNAs) are currently being evaluated as biomarkers of DIPI that may outperform and/or add value to the interpretation of amylase and lipase. To assess the relative performance of these novel miRNAs, a comprehensive evaluation was conducted to determine the sensitivity and specificity of detecting DIPI in rats. Four miRNAs were evaluated (miR-216a-5p, miR-216b-5p, miR-217-5p, and miR-375-3p) in plasma from 10 studies in which rats were treated with known pancreatic toxicants to assess sensitivity, and from 10 different studies in which toxicity was evident in tissues other than pancreas to assess specificity. The candidate miRNA biomarker performance was compared with amylase and lipase, and receiver operator characteristics (ROC) were determined. Analysis of ROCs demonstrated that all four miRNAs outperformed amylase and lipase in monitoring acute pancreatic injury defined as acinar cell degeneration/necrosis. Specifically, miR-217-5p had the highest performance among all biomarkers assessed. The increased sensitivity and specificity of these miRNAs support their use as biomarkers of DIPI, thereby adding value to the interpretation of amylase and lipase measurements in nonclinical studies. The potential for miRNAs to serve as translational biomarkers in the clinic for the monitoring of DIPI is also supported by this investigation.

Liver and skeletal muscle-specific microRNAs (miRNAs) are currently being evaluated as novel plasma biomarkers that may out-perform or add value to the conventional liver injury biomarkers alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and to the skeletal muscle injury biomarkers AST and creatine kinase (CK). A comprehensive evaluation was conducted to assess the relative performance of these miRNAs to detect and distinguish liver from muscle tissue injury. The performance of miR-122 and miR-192 for liver and miR-1, miR-133a, miR-133b, and miR-206 for skeletal muscle was compared with 10 enzymatic or protein biomarkers across 27 compounds causing specific types of tissue injury in rat. Receiver operator characteristic analyses were performed comparing the relative sensitivity and specificity of each of the biomarkers in individual animals with histopathology observations of necrosis and/or degeneration in various organs. All of the miRNAs outperformed ALT, AST, and/or CK in studies with either liver or skeletal muscle injury and demonstrated superior specificity in organs without type-specific injury (eg, liver biomarkers assessed with compounds that cause skeletal muscle injury). When additional protein biomarkers were included, glutamate dehydrogenase, arginase I, alpha-glutathione S-transferase for liver and skeletal troponin I, myosin light chain 3, fatty acid-binding protein 3, and creatine kinase M isoform for skeletal muscle, the miRNAs demonstrated equal or superior performance to the extended panel. Taken together, this comprehensive evaluation demonstrates that these novel miRNA toxicity biomarkers outperform and add value with respect to sensitivity and specificity over ALT, AST in monitoring the liver and over CK for monitoring skeletal muscle drug-induced injury.

As part of the Japanese Center for the Validation of Alternative Methods (JaCVAM) initiative international validation study of the in vivo rat alkaline comet assay (comet assay), we examined the ability of the assay to determine the genotoxicity of 2-acetylaminofluorene (AAF), azidothymidine (AZT), cisplatin (CPN), and isobutyraldehyde (IBA) in liver and glandular stomach of male Sprague-Dawley rats. Rats were given oral doses of test compound or control once daily for three days. High dose levels were approximately maximum tolerated doses and were based on preliminary range-finding studies. Tissues were harvested 3h after the final dose (48h after the initial dose). A bone marrow micronucleus assay (MN) was also conducted on the rats treated with AZT, CPN, and IBA. Acute toxic effects of treatment were determined primarily through histomorphologic analysis of liver and stomach but also by body weight and serum liver enzyme changes. The comet assay was conducted on fresh tissue preparations but frozen samples from two studies were also assayed. Statistically significant dose-related differences in comet % DNA in tail were found in liver and stomach for the genotoxin AZT and in liver for the genotoxin CPN, but not in liver or stomach for the non-genotoxin IBA. Statistically significant differences in % DNA in tail were measured in liver for the low and mid dose of the genotoxin AAF, but not the high dose. The comet assays of frozen liver suspensions from CPN- and AAF-treated rats yielded comparable results to the assays of fresh preparations. There were no indications of significant toxicity induced by any treatment. The micronucleus assay was positive for CPN and AZT and negative for IBA. In conclusion, the in vivo comet assay is capable of detecting genotoxic effects of a variety of chemicals and may fill an important role in the genotoxicity test battery.

Rodents, mice and rats in particular, are the species of choice for evaluating chemical carcinogenesis. However, different species and strains often respond very differently, undermining the logic of extrapolation of animal results to humans and complicating risk assessment. Intracisternal A particles (IAPs), endogenous retroviral sequences, are an important class of transposable elements that induce genomic mutations and cell transformation by disrupting gene expression. Several lines of evidence support a role of IAPs as mouse-specific genetic factors in responses to toxicity and expression of disease phenotypes. Since multiple subtypes and copies of IAPs are present in the mouse genome, their activity and locations relative to functional genes are of critical importance. This study identified the major "active" subtypes of IAPs (subtype 1/1a) that are responsible for newly transposed IAP insertions described in the literature, and confirmed that (1) polymorphisms for IAP insertions exist among different mouse strains and (2) promoter activity of the LTRs can be modulated by chemicals. This study further identified all the genes in the C57BL/6 mouse genome with IAP subtype 1 and 1a sequences inserted in their proximity, and the major biofunctional categories and cellular signaling networks of those genes. Since many "IAP-associated genes" play important roles in the regulation of cell proliferation, cell cycle, and cell death, the associated IAPs, upon activation, can affect cellular responses to xenobiotics and disease processes, especially carcinogenesis. This systemic analysis provides a solid foundation for further investigations of the role of IAPs as species- and strain-specific disease susceptibility factors.

2115 We evaluated the potential genotoxicity risk of 4-trifluoromethylaniline (TFMA), a material used in the synthesis of pharmaceutical drugs and a known structural component and metabolite of a marketed drug, in various short-term in vitro and in vivo test systems. TFMA increased mutations 5.4- to 14-fold (maximum) over background levels at 1000 or 3000 μg/plate in Salmonella typhimurium strains TA100 and TA1535 and in an E. Coli strain WP2 uvrA pKM101, both with and without S9 activation in a plate incorporation assay. In a pre-incubation assay in TA100, 2.1- to 3.7-fold increases in mutation were seen and DNA adducts were found at 100 to 1000 μg/plate (equivalent to 0.9 to 8.9 mM) both with and without S9 activation. Adducts were measured by the 32P-postlabeling assay, using nuclease P1 and butanol for enhancement with HPLC and TLC for separation. Both mutagenic and adduct signals were slightly ( 90%) and some minor adducts were seen, which were chromatographically similar in bacteria, hepatocytes, and calf thymus DNA with and without S9. The numbers of adducts per 106 nucleotides were in the range 0.01 to 0.2 in bacteria and hepatocytes and 3 to 9 in calf thymus DNA at the concentrations tested. On a per mM basis, the order of adduct response was bacteria

1895 The extent of incorporation of exogenously administered bromodeoxyuridine (BrdU) into DNA is an indicator of the rate of DNA synthesis and cell proliferation. We developed and validated an HPLC method for quantification of BrdU in DNA, based on the separation of nucleosides and their detection by an online UV detector. The baseline separation of BrdU from both normal deoxyribonucleosides and ribonucleosides was achieved in a 60-min run on a reversed-phased C18 column, using a methanol/ammonium formate buffer, as the eluent. The limit of detection was about 0.01 nmole BrdU using a standard BrdU solution. DNA was enzymatically hydrolyzed to deoxyribonucleosides prior to HPLC. The method allowed the detection of background levels of BrdU incorporation into DNA (45 μg) of urinary bladder and duodenum of control rats after a continuous administration of a BrdU solution (50 mg/ml) at a flow rate of 10 μl/h for 4 d, using a subcutaneously implanted osmatic minipump. Compared with the levels obtained by this continuous dosing, BrdU incorporation was much lower in rats dosed intraperitoneally (IP) 120 mg/kg BrdU at 19 h and 4 h prior to sacrifice. A substantial increase (about 10-fold) in BrdU incorporation was seen in the urinary bladder DNA of rats fed a diet containing 3% uracil for 4 d, as compared with the corresponding values obtained for rats fed normal rodent diet (Purina® Chow). BrdU incorporation was much higher (about 7-fold) using the continuous dosing regimen than the two-IP injection regimen. These results were consistent with those obtained by the immunohistochemical staining of BrdU-labeled nuclei with anti-BrdU antibody. Feeding rats 3% uracil in the diet rapidly induces urinary calculi and bladder hyperplasia, which progresses with continued treatment to papillomatosis and transitional cell carcinoma. An HPLC-based 32P-postlabeling assay was also developed for the measurement of BrdU in small amounts of DNA (1 μg). 32P-labeled nucleotides were prepared as the monophosphates, resolved using a 20-min run on a reversed-phase column with a methanol/ammonium formate buffer, and detected using an online radiochemical detector. The assay had similar sensitivity to the UV/HPLC method using about 5 μg DNA, but the latter method allowed the use of more DNA, resulting in higher sensitivity. In conclusion, we developed and validated HPLC/UV and HPLC/32P-postlabeling methods for the quantification of BrdU in DNA and demonstrated their application to quantify the incorporation of BrdU into DNA of rat tissues.

3-Methylindole (3MI), melatonin (Mel), serotonin (Ser), and tryptamine (Tryp) were evaluated in vitro for their potential to induce DNA adducts, DNA strand breaks, chromosomal aberrations (Abs), inhibition of DNA synthesis, and mutations. All compounds produced DNA adducts in calf thymus DNA in the presence of rat liver S9. In cultured rat hepatocytes, all produced DNA adducts but none induced DNA strand breaks. In Chinese hamster ovary cells, 3MI and Mel produced DNA adducts, Abs, and inhibition of DNA synthesis with and without S9, except that Mel without S9 did not form adducts. Ser formed DNA adducts, was an equivocal Abs inducer, and suppressed DNA synthesis. Tryp induced neither adducts nor Abs, but did suppress DNA synthesis with S9. Ser and Tryp were less cytotoxic than 3MI and Mel. Mel, Ser, and Tryp failed to induce mutations in Salmonella and E. coli strains with or without S9. 3MI and Mel produced DNA adducts but not mutations in Salmonella TA100 with S9. 3MI and its metabolite indole 3-carbinol also did not induce mutations in a shuttle vector system in human cells. The lack of correlation between DNA adducts and other genotoxicity endpoints for these indole compounds may be due to the higher sensitivity of the (32)P-postlabeling adduct assay or it may indicate that the indole-DNA adducts per se are not mutagenic and are not able to induce strand breaks or alkali-labile lesions. The indole-induced Abs may result from cytotoxicity and suppression of DNA synthesis with minimal if any contribution from DNA adducts.

The in vitro alkaline elution/rat hepatocyte assay is a sensitive assay for genotoxicity, measured as DNA strand breaks induced in primary cultures of rat hepatocytes after 3-h treatments with test compounds. Since DNA degradation can be rapid and extensive in dead and/or dying cells, the original criteria for a positive result in the assay were that a compound induce a 3.0-fold or greater increase in the elution slope (for the terminal phase of alkaline elution from 3 to 9 h) in the absence of significant cytotoxicity (defined as relative cell viability of less than 70% by trypan blue dye exclusion; TBDE). Recently we have shown that false-positive results can still be obtained due to cytotoxicity when loss of membrane integrity is a late event in toxic cell death relative to the induction of endonucleolytic DNA degradation. To improve the ability of the assay to discriminate between genotoxic vs. cytotoxic effects of chemicals, we have evaluated additional assays of cytotoxicity including cell adenosine triphosphate (ATP) and potassium (K+) content, tetrazolium dye reduction (MTT), TBDE after a further 3-h recovery incubation without test chemicals (delayed toxicity), cell blebbing and endonucleolytic DNA degradation (double-strand breaks; DSBs) assessed by pulsed-field gel electrophoresis (PFGE). We have also evaluated 2 parameters derived from the elution data which can indicate extensive, cytotoxicity-induced DNA degradation: the fraction of the DNA recovered in the neutral lysis/rinse fraction and the gamma-intercept of the extrapolation of the 3-9-h segment of the elution curve. Twenty-eight rodent non-carcinogens that are negative (or inconclusive) in the Ames assay with no, or limited, other evidence of genotoxicity, and 33 genotoxins, most of which are also carcinogens, were evaluated. The results showed that DNA degradation as measured by a 1-h PACE (Programmed Autonomously Controlled Electrodes)/PFGE assay was a sensitive indicator of cytotoxicity which correlated well with results of the other cytotoxicity indicators. The delayed TBDE (after a 3-h recovery), intracellular potassium and ATP assays as well as the gamma-intercept parameter were also shown to be sensitive and in some cases complementary measures of cytotoxicity. Using new criteria based on these data of an induced slope (treatment slope-negative control slope) of 0.020 for the 3- to 9-h elution period and cytotoxicity limits of 70% relative viability for the delayed TBDE assay and 50% for intracellular ATP content, the assay scores the genotoxicity of these 61 reference compounds with an overall accuracy of 92%. Test results using these new criteria are provided for an additional 20 compounds (5 non-genotoxic carcinogens and 15 compounds whose genotoxic and carcinogenic potential are unknown or equivocal).

The use of genetic toxicology tests for hazard identification is complicated by the fact that some in vitro tests using cultured mammalian cells are subject to potential artifacts that can make it difficult to distinguish between direct, chemically-induced genotoxicity, and DNA damage that occurs secondary to chemically-induced cytotoxicity (e.g., mediated by endogenous nucleases). Recently, we demonstrated that cytotoxicity-induced DNA double strand breaks (dsb) can produce artifacts in the in vitro alkaline elution/rat hepatocyte assay [Elia et al., 1993]. To explore this further, we used pulsed field gel/DNA dsb assays to characterize the relationship between chemically-induced cytotoxicity and the degradation of genomic DNA to high molecular weight fragments. Two sets of compounds were tested: 17 cytotoxic agents judged to be neither genotoxic nor carcinogenic, and 10 known genotoxic carcinogens. We found a close correlation between chemically-induced cytotoxicity and the rapid degradation of DNA to high molecular weight, double-stranded fragments. In contrast, the classic genotoxic chemicals tested generally did not trigger DNA dsb fragmentation at doses that were genotoxic but not immediately cytotoxic. These data indicate that pulsed field gel/DNA dsb assays can be used with in vitro genetic toxicology assays to help distinguish between genotoxic and cytotoxic mechanisms of DNA damage. (C) 1994 Wiley-Liss, Inc.