Bacterial mutagenicity screening in the pharmaceutical industry.

Genetic toxicity testing is used as an early surrogate for carcinogenicity testing. Genetic toxicity testing is also required by regulatory agencies to be conducted prior to initiation of first in human clinical trials and subsequent marketing for most small molecule pharmaceutical compounds. To reduce the chances of advancing mutagenic pharmaceutical candidates through the drug discovery and development processes, companies have focused on developing testing strategies to maximize hazard identification while minimizing resource expenditure due to late stage attrition. With a large number of testing options, consensus has not been reached on the best mutagenicity platform to use or on the best time to use a specific test to aid in the selection of drug candidates for development. Most companies use a process in which compounds are initially screened for mutagenicity early in drug development using tests that require only a few milligrams of compound and then follow those studies up with a more robust mutagenicity test prior to selecting a compound for full development. This review summarizes the current applications of bacterial mutagenicity assays utilized by pharmaceutical companies in early and late discovery programs. The initial impetus for this review was derived from a workshop on bacterial mutagenicity screening in the pharmaceutical industry presented at the 40th Annual Environmental Mutagen Society Meeting held in St. Louis, MO in October, 2009. However, included in this review are succinct summaries of use and interpretation of genetic toxicity assays, several mutagenicity assays that were not presented at the meeting, and updates to testing strategies resulting in current state-of the art description of best practices. In addition, here we discuss the advantages and liabilities of many broadly used mutagenicity screening platforms and strategies used by pharmaceutical companies. The sensitivity and specificity of these early mutagenicity screening assays using proprietary compounds and their concordance (predictivity) with the regulatory bacterial mutation test are discussed.

The radiation-induced bystander effect for clonogenic survival.

It has long been accepted that the radiation-induced heritable effects in mammalian cells are the result of direct DNA damage. Recent evidence, however, suggests that when a cell population is exposed to a low dose of alpha particles, biological effects occur in a larger proportion of cells than are estimated to have been traversed by alpha particles. Experiments involving the Columbia University microbeam, which allows a known fraction of cells to be traversed by a defined number of alpha particles, have demonstrated a bystander effect for clonogenic survival and oncogenic transformation in C3H 10T(1/2) cells. When 1 to 16 alpha particles were passed through the nuclei of 10% of a C3H 10T(1/2) cell population, more cells were unable to form colonies than were actually traversed by alpha particles. Both hit and non-hit cells contributed to the outcome of the experiments. The present work was undertaken to assess the bystander effect of radiation in only non-hit cells. For this purpose, Chinese hamster V79 cells transfected with hygromycin- or neomycin-resistance genes were used. V79 cells stably transfected with a hygromycin resistance gene and stained with a nuclear dye were irradiated with the charged-particle microbeam in the presence of neomycin-resistant cells. The biological effect was studied in the neomycin-resistant V79 cells after selective removal of the hit cells with geneticin treatment.

Routine screening mammography: how important is the radiation-risk side of the benefit-risk equation?

The potential radiation hazards associated with routine screening mammography, in terms of breast cancer induction, are discussed in the context of the potential benefits. The very low energy X-rays used in screening mammography (26-30 kVp) are expected to be more hazardous, per unit dose, than high-energy X- or gamma-rays, such as those to which A-bomb survivors (from which radiation risk estimates are derived) were exposed. Based on in vitro studies using oncogenic transformation and chromosome aberration end-points, as well as theoretical estimates, it seems likely that low doses of low-energy X-rays produce an increased risk per unit dose (compared with high energy photons) of about a factor of 2. Because of the low doses involved in screening mammography, the benefit-risk ratio for older women would still be expected to be large, though for younger women the increase in the estimated radiation risk suggests a somewhat later age than currently recommended--by about 5-10 years--at which to commence routine breast screening.

The role of adrenal corticosteroids in induction of micronuclei by morphine.

It has previously been shown that morphine can increase the frequency of micronucleated splenocytes when administered to mice, but not when cells are exposed to the opiate in vitro. Morphine treatment is also known to increase circulating levels of glucocorticosteroids, which have been reported to produce genetic damage in vivo and in vitro. In order to determine whether adrenal hormones might mediate the genotoxic effects of morphine, adrenalectomized and sham-operated mice were treated with morphine sulfate. In sham-operated animals administration of morphine produced a dose-related increase in the frequency of micronucleated cells, whereas adrenalectomy abolished the effect. When plasma from morphine-treated mice was used to supplement growth medium of untreated splenocytes, the frequency of micronucleated cells increased, an effect partially blocked by the steroid antagonist RU 486. The N-methylmorphine, which does not stimulate the release of corticosterone from adrenal glands, induced micronuclei formation in splenocytes, and administration of metyrapone, an inhibitor of corticosterone biosynthesis, blocked the morphine-induced increase in corticosterone secretion, but had no effect on the frequency of micronuclei formation. These results indicate that basal levels of glucocorticosteroids are required for induction of micronuclei by morphine in murine splenocytes, but activation of the hypothalamo-pituitary-adrenal (HPA) axis by morphine does not contribute to the observed response.

Adaptive response and the bystander effect induced by radiation in C3H 10T(1/2) cells in culture.

This paper discusses two phenomena of importance at low doses that have an impact on the shape of the dose-response relationship. First, there is the bystander effect, the term used to describe the biological effects observed in cells that are not themselves traversed by a charged particle, but are neighbors of cells that are; this exaggerates the effect of small doses of radiation. Second, there is the adaptive response, whereby exposure to a low level of DNA stress renders cells resistant to a subsequent exposure; this reduces the effect of low doses of radiation. The present work was undertaken to assess the relative importance of the adaptive response and the bystander effect induced by radiation in C3H 10T(1/2) cells in culture. When the single-cell microbeam delivered from 1 to 12 alpha particles through the nuclei of 10% of C3H 10T(1/2) cells, more cells were inactivated than were actually traversed by alpha particles. The magnitude of this bystander effect increased with the number of particles per cell. An adaptive dose of 2 cGy of gamma rays, delivered 6 h beforehand, canceled out about half of the bystander effect produced by the alpha particles.

The bystander effect in radiation oncogenesis: I. Transformation in C3H 10T1/2 cells in vitro can be initiated in the unirradiated neighbors of irradiated cells.

It has long been accepted that radiation-induced genetic effects require that DNA be hit and damaged directly by the radiation. Recently, evidence has accumulated that in cell populations exposed to low doses of alpha particles, biological effects occur in a larger proportion of cells than are estimated to have been traversed by alpha particles. The end points observed include chromosome aberrations, mutations and gene expression. The development of a fast single-cell microbeam now makes it possible to expose a precisely known proportion of cells in a population to exactly defined numbers of alpha particles, and to assay for oncogenic transformation. The single-cell microbeam delivered no, one, two, four or eight alpha particles through the nuclei of all or just 10% of C3H 10T1/2 cells. We show that (a) more cells can be inactivated than were actually traversed by alpha particles and (b) when 10% of the cells on a dish are exposed to alpha particles, the resulting frequency of induced transformation is not less than that observed when every cell on the dish is exposed to the same number of alpha particles. These observations constitute evidence suggesting a bystander effect, i.e., that unirradiated cells are responding to damage induced in irradiated cells. This bystander effect in a biological system of relevance to carcinogenesis could have significant implications for risk estimation for low-dose radiation.

Atm inactivation results in aberrant telomere clustering during meiotic prophase.

A-T (ataxia telangiectasia) individuals frequently display gonadal atrophy, and Atm-/- mice show spermatogenic failure due to arrest at prophase of meiosis I. Chromosomal movements take place during meiotic prophase, with telomeres congregating on the nuclear envelope to transiently form a cluster during the leptotene/zygotene transition (bouquet arrangement). Since the ATM protein has been implicated in telomere metabolism of somatic cells, we have set out to investigate the effects of Atm inactivation on meiotic telomere behavior. Fluorescent in situ hybridization and synaptonemal complex (SC) immunostaining of structurally preserved spermatocytes I revealed that telomere clustering occurs aberrantly in Atm-/- mice. Numerous spermatocytes of Atm-/- mice displayed locally accumulated telomeres with stretches of SC near the clustered chromosome ends. This contrasted with spermatogenesis of normal mice, where only a few leptotene/zygotene spermatocytes I with clustered telomeres were detected. Pachytene nuclei, which were much more abundant in normal mice, displayed telomeres scattered over the nuclear periphery. It appears that the timing and occurrence of chromosome polarization is altered in Atm-/- mice. When we examined telomere-nuclear matrix interactions in spermatocytes I, a significant difference was observed in the ratio of soluble versus matrix-associated telomeric DNA sequences between meiocytes of Atm-/- and control mice. We propose that the severe disruption of spermatogenesis during early prophase I in the absence of functional Atm may be partly due to altered interactions of telomeres with the nuclear matrix and distorted meiotic telomere clustering.

Telomerase activity as a measure for monitoring radiocurability of tumor cells.

Radiotherapy plays a key role in the treatment of many tumors. It is difficult to determine what fraction of tumor cells survives after treatment with ionizing radiation. A convenient and sensitive biochemical assay could be efficacious in determining the potential success of radiotherapy. Since telomerase activity is frequently associated with the malignant phenotype, we sought to determine whether a correlation existed between ionizing radiation-induced cell killing and telomerase activity. We evaluated telomerase activity in two telomerase-positive and one telomerase-negative human cell line exposed to ionizing radiation. Telomerase activity was determined using a PCR-based telomeric repeat amplification protocol coupled with ELISA. We found ionizing radiation treatment to decrease the telomerase activity (in plateau phase cells of RKO, HeLa; and growing cells of RKO) in a dose-dependent manner, which correlated with cell death in in vitro tests as well as during tumor regression in nude mice. In contrast, growing HeLa cells after 24 h postradiation treatment showed an increase in telomerase activity, but there was no increase in the levels of mRNA of hTERT. To assess the sensitivity of the telomerase activity assay, we performed mixing experiments of HeLa and AG1522 cell extracts. These studies showed that telomerase activity could be detected in lysate equal to a single HeLa cell when mixed with 10,000 AG1522 cells. Our results indicate that even a few surviving neoplastic cells can be detected by telomerase activity assay. Therefore, detection of telomerase activity may be a useful monitor of radiotherapeutic efficacy and an early predictor of outcome.

Penclomedine-induced DNA fragmentation and p53 accumulation correlate with reproductive cell death in colorectal carcinoma cells with altered p53 status.

Penclomedine, a synthetic pyridine derivative, has documented antitumor activity and is being investigated in clinical trials. Its mechanism of action is unknown although it may be metabolized to a free radical, DNA-reactive species. We previously reported that telomerase positive colorectal carcinoma (RKO) cells with abrogated p53 function were more sensitive to penclomedine than were telomerase positive cells with wild-type p53. The present study demonstrates that significant differences in DNA fragmentation in response to penclomedine were observed in RKO cells lacking functional p53 compared with RKO cells with normal p53 function. No differences in DNA fragmentation in response to ionizing radiation were seen in RKO cells with normal or abrogated p53 function. RKO cells with functional p53 respond to penclomedine treatment with a dose-dependent increase in p53 protein levels. However, RKO cells with abrogated p53 function did not show any such change in p53 protein levels. Further, p53-independent increase of p21 was observed, although the significance of this response remains uncertain. These studies suggest that penclomedine may have a therapeutic advantage in killing cells that have abrogated p53 function.

A preliminary report: frequency of A-T heterozygotes among prostate cancer patients with severe late responses to radiation therapy.

PURPOSE: To investigate whether a significant proportion of prostate cancer patients who have late sequelae after high-dose external-beam conformal radiation therapy are radio-sensitive because they are carriers of ataxia-telangiectasia, that is, are heterozygous for mutations in the ATM gene. PATIENTS AND METHODS: A group of prostate cancer patients were selected who experienced severe late sequelae, specifically proctitis or cystitis, after high-dose external-beam conformal radiation therapy, together with a control group of patients treated in the same way but who did not have severe late effects. Blood samples were taken from these patients, genomic DNA extracted, and mutations sought in the ATM gene. RESULTS: Of 17 late-effect patients in whom most or all of the ATM gene has been examined, significant mutations (17.6%) were identified in three. No significant mutations were found in the control group. The incidence of ataxia- telangiectasia heterozygotes in the United States population is 1% to 2%. DISCUSSION: These preliminary data suggest that a disproportionate number, but by no means all, of prostate cancer radiotherapy patients who experience severe late effects are ataxia-telangiectasia heterozygotes. If this conclusion is confirmed, these individuals could be identified prospectively and, with dose de-escalation, spared a great deal of discomfort and suffering. As a corollary, if most of the small late-effects population were prospectively identifiable, the dose to the remaining population could potentially be escalated. Present methods of identifying mutations in a large gene, such as ATM, are cumbersome and expensive, but the technology is evolving rapidly, so that rapid screening of the ATM gene is imminent.

Molecular cloning and chromosomal localization of Chinese hamster telomeric protein chTRF1. Its potential role in chromosomal instability.

Chinese hamster cells frequently have altered karyotypes. To investigate the basis of recent observations that karyotypic alterations are related to telomeric fusions, we asked whether these alterations are due to lack of telomere repeat binding factor/s. Further, Chinese hamster chromosomes contain large blocks of interstitial telomeric repeats, which are preferentially involved in chromosome breakage and exchange, rendering it an interesting model for such studies. Here, we report on the cloning and the chromosomal localization of the Chinese hamster telomere repeat binding factor, chTRF1. The sequence analysis revealed, similar to human TRF1 (hTRF1), an N-terminal acidic domain, a TRF1 specific DNA binding motif and a C-terminal Myb type domain. Unlike mouse TRF1 (mTRF1), chTRF1 shows 97.5% identity to hTRF1. chTRF1 gene was localized on the long arm of chromosome 5. In vitro translation of chTRF1 resulted in protein product similar in molecular weight to hTRF1. Immunostaining of Chinese hamster ovary cells (CHO) with anti-TRF1 antibody revealed punctate nuclear staining. At metaphase, antibodies failed to detect TRF1 on most of the chromosome ends and the interstitial telomeric repeat bands. These studies suggest that chTRF1 does not bind the interstitial telomeric repeats, and its presence at the metaphase chromosome ends is limited. The later could be a factor contributing to frequent karyotypic alterations observed in Chinese hamster cells.

Influence of ATM function on telomere metabolism.

The ATM gene product, which is defective in the cancer-prone disorder ataxia telangiectasia, has been implicated in mitogenic signal transduction, chromosome condensation, meiotic recombination and cell cycle control. The ATM gene has homology with the TEL1 gene of yeast, mutations of which lead to shortened telomeres. To test the hypothesis that the ATM gene product is involved in telomere metabolism, we examined telomeric associations (TA), telomere length, and telomerase activity in human cells expressing either dominant-negative or complementing fragments of the ATM gene. The phenotype of RKO colorectal tumor cells expressing ATM fragments containing a leucine zipper (LZ) motif mimics that of ataxia telangiectasia (A-T) cells. These transfected RKO cells relative to transfected controls had a higher frequency of cells with TA and shortened telomeres, but no detectable change in telomerase activity. In addition, the percentage of cells with TA after gamma irradiation was higher in the transfected RKO cells with dominant negative activity of the ATM gene, compared to control cells. SV40 transformed fibroblasts derived from an A-T patient and transfected with a complementing carboxyl terminal kinase region of the ATM gene had a reduced frequency of cells with TA, with no effect on the telomere length or telomerase activity. The present studies using isogenic cells with manipulated ATM function demonstrate a role for the ATM gene product in telomere metabolism.

Continuous Measurement of Changes in Intracellular Calcium Concentration in Mouse Splenic T Cells Attached to a Glass Substrate.

Mitogen- and isoproterenol-induced changes of [Ca(2+)](i) in T cells attached to a glass substrate were examined. Murine (C57BL/6) splenic T cells were attached to coverslips or 35-mm dishes (MatTek) precoated with Cell Tak((R)) (3.5 &mgr;g/cm(2)). The cells were then loaded with fluorescent dye (2 &mgr;g/ml of fura2-AM or fluo3-AM) and changes in [Ca(2+)](i) in a population of cells (using a spectrofluorometer) or in single cells (using a confocal microscope) were measured during continuous superfusion. Population measurements of [Ca(2+)](i) demonstrated that concanavalin A (Con A, 2 or 5 &mgr;g/ml) caused an increase in [Ca(2+)](i) that rose to a peak and then declined to a steady state. The concentration-response relationship (0.05-5 &mgr;g/ml) had an EC(50) of approximately 0.3 &mgr;g/ml. Isoproterenol decreased the Con A-induced elevation of steady state [Ca(2+)](i). In single cell studies, the increase in [Ca(2+)](i) in response to Con A typically occurred in about 50% of the cells in a microscope field, and the delay before activation varied among cells. Taken together these data demonstrate that Cell Tak((R)) can be used to attach T cells to glass coverslips and will be useful for the study of signaling mechanisms in T cells. Copyright 1995 S. Karger AG, Basel

The clastogenicity of morphine sulfate in vivo.

An opioid analgesic, morphine, and an opioid peptide, beta-endorphin, have been shown to induce chromosome damage, as indicated by an increased frequency of micronucleated lymphocytes, following acute administration to mice. The genotoxic response is opioid receptor-mediated and is abolished in adrenalectomized animals. Further, plasma from morphine-treated animals also induces micronuclei formation in naive lymphocytes in vitro; this response is blocked by inclusion the steroid antagonist RU 486 in the incubation mixture. In addition to the steroid-mediated production of chromosome damage, morphine acts directly on lymphocytes to enhance the clastogenicity of acutely administered cyclophosphamide in manner consistent with depressed DNA repair capacity.

Induction of micronuclei in murine lymphocytes by morphine.

Although individuals who abuse drugs are prone to an increased risk of malignancy, the mutagenic and carcinogenic potential of these agents has received relatively little attention. We report here on the potential of morphine to induce micronuclei in murine lymphocytes. Following a single intraperitoneal injection of 20 mg/kg morphine, the frequency of micronucleated binuclear (cytochalasin-blocked) murine T- and B-splenocytes was elevated from 12-36 hr after treatment. The maximum frequencies seen 24 hr after injection were 6.3- and 4.9-fold greater than the respective controls. A dose-dependent induction of micronuclei was observed from 5-20 mg/kg morphine, with no further increases in frequency produced by higher doses. In contrast, incubation of mitogen-stimulated splenocytes with 10(-7)-10(-4) M morphine in vitro produced no change in frequency of micronucleated cells relative to controls. Treatment with the narcotic antagonist naloxone (5 mg/kg) alone had no effect on the frequency of micronuclei, but reduced the clastogenic response of a subsequently administered dose of morphine (20 mg/kg). Thus, in murine lymphocytes morphine indirectly produces genetic damage, which is at least in part opioid receptor-mediated.

Toxicity of dietary monensin in quail.

Quail were fed monensin to determine liver damage, as measured by changes in activities of serum enzymes and liver microsomal enzymes. Monensin fed at a therapeutic level of 110 ppm for 2 weeks produced an increase in cytochrome P-450 and cytochrome b5 and induction of the activities of benzphetamine N-demethylase, aminopyrine N-demethylase, and aniline hydroxylase, with no changes in the activities of serum sorbitol dehydrogenase (SDH), alanine aminotransferase (ALT), and aspartate aminotransferase (AST). On the other hand, quail fed 110 ppm, 220 ppm, and 330 ppm monensin in feed for 6 weeks showed a significant rise in SDH and AST activities at 330 ppm but not at 110 ppm and 220 ppm. The manifestations of liver toxicity observed at 330 ppm were accompanied by a significant decrease in all the aforementioned hepatic microsomal mixed-function oxidases. In contrast, quail fed monensin at 110 ppm and 220 ppm for 6 weeks produced no change in these parameters except for benzphetamine N-demethylase, aminopyrine N-demethylase, and aniline hydroxylase, which were significantly increased in birds fed 220 ppm of monensin.

Studies on monensin toxicity in goats.

This study was carried out to examine the effect of orally administered monensin on the liver of goats. Goats given monensin at the rate of 55 mg/kg of feed (55 ppm) for three weeks showed clinical signs of anorexia and diarrhea, along with increased pentobarbital sleeping time and serum SDH level indicating the possible presence of hepatotoxicity. On the other hand, animals fed monensin at 11 ppm for three weeks or 8 mg/kg of body weight/day for five days showed no signs of hepatotoxicity.