Evidence for the contribution of non-covalent steroid interactions between DNA and topoisomerase in the genotoxicity of steroids.

Fifty two steroids and 9 Vitamin D analogs were docked into ten crystallographically-defined DNA dinucleotide sites and two human topoisomerase II ATP binding sites using two computational programs, Autodock and Surflex. It is shown that both steroids and Vitamin D analogs exhibit a propensity for non-covalent intercalative binding to DNA. A higher predicted binding affinity was found, however, for steroids and the ATP binding site of topoisomerase; in fact these drugs exhibited among the highest topo II binding observed in over 1370 docked drugs. These findings along with genotoxicity data from 26 additional steroids not subjected to docking analysis, support a mechanism wherein the long known, but poorly understood, clastogenicity of steroids may be attributable to inhibition of topoisomerase. A "proof of principle" experiment with dexamethasone demonstrated this to be the likely mechanism of clastogenicity of, at least, this steroid. The generality of this proposed mechanism of genotoxicity across the steroids and vitamin-D analogs is discussed.

Circulating markers of vascular injury and angiogenesis in antineutrophil cytoplasmic antibody-associated vasculitis.

OBJECTIVE: To identify biomarkers that distinguish between active antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) and remission in a manner superior or complementary to established markers of systemic inflammation. METHODS: Markers of vascular injury and angiogenesis were measured before and after treatment in a large clinical trial in AAV: 163 subjects enrolled in the Rituximab in ANCA-Associated Vasculitis trial were screened for the present study. Serum levels of E-selectin, intercellular adhesion molecule 3 matrix metalloproteinase protein 1 (MMP-1), MMP-3, MMP-9, P-selectin, thrombomodulin, and vascular endothelial growth factor were measured at study screening (time of active disease) and at month 6. Erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) levels had been measured at the time of the clinical visit. The primary outcome measure was the difference in marker level between screening and month 6 among patients whose disease was in remission (Birmingham Vasculitis Activity Score for Wegener's granulomatosis [BVAS/WG] score of 0) at month 6. RESULTS: All patients had severe active vasculitis at screening (mean ± SD BVAS/WG score 8.6 ± 3.2). Among the 123 patients whose disease was clinically in remission at month 6, levels of all markers except E-selectin showed significant declines. MMP-3 levels were also higher among the 23 patients with active disease at month 6 than among the 123 patients whose disease was in remission. MMP-3 levels correlated weakly with ESR and CRP levels. CONCLUSION: Many markers of vascular injury and angiogenesis are elevated in severe active AAV and decline with treatment, but MMP-3 appears to distinguish active AAV from remission better than the other markers studied. Further study of MMP-3 is warranted to determine its clinical utility in combination with conventional markers of inflammation and ANCA titers.

Early events in vascular injury in the rat induced by the phosphodiesterase IV inhibitor SCH 351591.

Treatment with drugs from multiple classes induces vascular injury with medial necrosis, hemorrhage, endothelial damage, and inflammation. Previous research has suggested early events might be occurring well in advance of the full lesions that appear forty-eight to seventy-two hours after dosing with SCH 351591, a PDE IV inhibitor. This study was performed to study early events in detail. Rats were dosed with 20 mg/kg of drug by gavage and sacrificed at times between fifteen and 240 minutes after dosing. Tissues were collected for histopathological analysis and gene expression studies. Serum was collected for biomarker analysis. The data from biomarker analysis showed a three-part response with an early phase that was maximal at fifteen to thirty minutes, a second phase from forty-five to 180 minutes, and the third phase that was starting to rise at four hours. The first phase included increases in lymphocytes, serum histamine, and serum nitrite. The second phase shows continued elevation of serum nitrite. The third phase was marked by an increase in serum GRO/CINC-1. At fifteen minutes, histopathology showed activation of mast cells, but not degranulation. Increases in endothelial activation and perivascular inflammatory cells were first apparent at thirty minutes and increased through 240 minutes.

Effects of the histamine H1 antagonist chlorcyclizine on rat fetal palate development.

BACKGROUND: The effects of histamine H1 antagonist chlorcyclizine on rat palate development were characterized following in utero exposure. METHODS: To identify the optimum dose for inducing cleft palate, pregnant rats were administered 30, 60, or 90 mg/kg chlorcyclizine on Gestation Days 11 to 14. Fetal palate gene expression was also assessed after 90 mg/kg chlorcyclizine at 8, 15 and 30 hours post-dose on Gestation Day 14 using microarray and qRT-PCR. RESULTS: Rats in the 60- and 90-mg/kg groups exhibited adverse clinical signs and body weight loss. Rats in the 90-mg/kg group also demonstrated increases in late resorptions and decreases in fetal weight. Effects in the low-dose group were limited to decreases in body weight gain. Fetal assessment on Gestation Day 21 revealed that findings were limited to the 60- and 90-mg/kg groups, and included cleft palate (80% of litters for both groups), high arched palate, small nose, micrognathia, high domed head, digits shortened/absent and small limb. The fetal incidence of cleft palate was higher at 90 mg/kg, thus this dose was selected to assess palate gene expression. The altered genes associated with chlorcyclizine-induced cleft palate included Wnt5a, Bmp2, Bmp4, Fgf10, Fgfr2, Msx1, and Insig1 but the magnitude of the change was relatively small (1.5- to 2-fold). CONCLUSIONS: Expression of several genes involved in palate, limb and digit development was altered in the fetal palate following in utero exposure to chlorcyclizine. The subtle perturbation and interplay of these genes may have profound effects on the dynamics of fetal palate development.

ABCG2-associated resistance to Hoechst 33342 and topotecan in a murine cell model with constitutive expression of side population characteristics.

Drug resistant tumor "side-populations," enriched in cancer stem cells and identified by reduced accumulation of Hoechst 33342 under ABCG2-mediated efflux, may compromise therapeutic outcome. Side-population cells have predicted resistance to minor groove ligands, including the DNA topoisomerase I poison topotecan. We have used a stable Hoechst 33342-resistant murine L cell system (HoeR415) to study resistance patterns, removing the need for SP isolation before microarray analysis of gene expression and the tracking of cell cycle dynamics and cytotoxicity. The majority of HoeR415 cells displayed a side-population phenotype comparable with that of the side-population resident in the ABCG2 over-expressing A549 lung cancer cell line. Photo-crosslinking showed direct protection against minor groove ligand residence on DNA, driven by ABCG2-mediated efflux and not arising from any binding competition with endogenous polyamines. The covalent minor-groove binding properties of the drug FCE24517 (tallimustine) prevented resistance suggesting a mechanism for overcoming SP-related drug resistance. Hoechst 33342-resistant murine cells showed lower but significant crossresistance to topotecan, again attributable to enhanced ABCG2 expression, enabling cells to evade S-phase arrest. Hoechst 33342/TPT-resistant cells showed limited ancillary gene expression changes that could modify cellular capacity to cope with chronic stress including over-expression of Aldh1a1 and Mgst1, but under-expression of Plk2 and Nnt. There was no evidence to link the putative stem cell marker ALDH1A1 with any augmentation of the TPT resistance phenotype. The study has implications for the patterns of drug resistance arising during tumor repopulation and the basal resistance to minor groove-binding drugs of tumor side-populations.

Lovastatin enhances the genotoxicity of doxorubicin in Chinese hamster V79 cells via noncovalent DNA binding.

The 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A reductase inhibitor, lovastatin (lova), has been reported to both sensitize to, and protect against, the toxic effects of the antitumor anthracycline doxorubicin (dox) in cellular and in vivo systems. The mechanism by which these effects occur has not yet been determined. In the present study, lova is shown to enhance the genotoxicity of dox in the V79 cell in vitro micronucleus assay and to do so, most likely, via noncovalent interaction with DNA adjacent to sites of dox binding. These studies confirm and extend the experimental evidence strongly suggesting the importance of noncovalent drug/DNA interactions in cellular responses to genotoxic stimuli.

Validation of putative genomic biomarkers of nephrotoxicity in rats.

Drug-induced renal injury is a common finding in the early preclinical phase of drug development. But the specific genes responding to renal injury remain poorly defined. Identification of drug-induced gene changes is critical to provide insights into molecular mechanisms and detection of renal damage. To identify genes associated with the development of drug-induced nephrotoxicity, a literature survey was conducted and a panel of 48 genes was selected based on gene expression changes in multiple published studies. Male Sprague-Dawley rats were dosed daily for 1, 3 or 5 days to the known nephrotoxicants gentamicin, bacitracin, vancomycin and cisplatin, or the known hepatotoxicants ketoconazole, 1-naphthyl isothiocyanate and 4,4-diaminodiphenylmethane. Histopathological evaluation and clinical chemistry revealed renal proximal tubular necrosis in rats treated with the nephrotoxicants, but not from those treated with the hepatotoxicants. RNA was extracted from the kidney, and RT-PCR was performed to evaluate expression profiles of the selected genes. Among the genes examined, 24 genes are confirmed to be highly induced or repressed in rats treated with nephrotoxicants; further investigation identified that 5 of the 24 genes were also altered by hepatotoxicants. These data led to the identification of a set of genomic biomarker candidates whose expression in kidney is selectively regulated only by nephrotoxicants. Among those genes displaying the highest expression changes specifically in nephrotoxicant-treated rats were kidney injury molecule 1 (Kim1), lipocalin 2 (Lcn2), and osteopontin (Spp1). The establishment of such a genomic marker set offers a new tool in our ongoing quest to monitor nephrotoxicity.

Histopathology of vascular injury in Sprague-Dawley rats treated with phosphodiesterase IV inhibitor SCH 351591 or SCH 534385.

Histopathological and immunohistochemical studies were conducted to characterize vascular injuries in rats treated with phosphodiesterase (PDE) IV inhibitors SCH 351591 or SCH 534385. Sprague-Dawley rats were administered PDE IV inhibitors by gavage at a range of doses and times. The two PDE IV inhibitors induced comparable levels of vascular injury, primarily in the mesentery and to a lesser extent in the pancreas, kidney, liver, small intestine, and stomach. Mesenteric vascular changes occurred as early as one hour, progressively developed over twenty-four to forty-eight hours, peaked at seventy-two hours, and gradually subsided from seven to nine days. The typical morphology of the vascular toxicity consisted of hemorrhage and necrosis of arterioles and arteries, microvascular injury, fibrin deposition, and perivascular inflammation of a variety of blood vessels. The incidence and severity of mesenteric vascular injury increased in a time- and dose-dependent manner in SCH 351591- or SCH 534385-treated rats. Mesenteric vascular injury was frequently associated with activation of mast cells (MC), endothelial cells (EC), and macrophages (MØ). Immunohistochemical studies showed increases in CD63 immunoreactivity of mesenteric MC and in nitrotyrosine immunoreactivity of mesenteric EC and MØ. The present study also provides a morphological and cellular basis for evaluating candidate biomarkers of drug-induced vascular injury.

Prediction of non-genotoxic carcinogenesis in rats using changes in gene expression following acute dosing.

Non-genotoxic carcinogenicity of chemicals is currently routinely evaluated in 2-year rodent bioassays. Therefore, the development of early biomarkers for non-genotoxic carcinogenesis would result in substantial savings in time and expense. The current study investigates whether early changes in gene expression may be developed as markers for cancer. Animals were treated for 1 or 5 days with either non-genotoxic carcinogens (NGTCs) or non-carcinogens and gene expression was analyzed by quantitative PCR (qPCR). We tested two gene signatures previously reported to detect non-genotoxic carcinogens. Using one gene signature it was confirmed that 3/3 non-genotoxic carcinogens and 2/2 non-carcinogens are correctly identified with data from 1 or 5 days of dosing. In contrast an alternative signature correctly identified 0/3 and 2/3 non-genotoxic carcinogens at 1 and 5 days of treatment, respectively and 2/2 non-carcinogens at both time-points. Additionally, we evaluated a novel panel of putative biomarker genes, from the literature, many of which have roles in cell growth and division, including myc, cdc2 and mcm6. These genes were significantly induced by non-genotoxic carcinogens and not by non-carcinogens. Using the average fold-induction across this panel, 2/3 non-genotoxic carcinogens were detected at both 1 and 5 days. These data support the idea that acute changes in gene expression may provide biomarkers for non-genotoxic carcinogenesis but also highlight interesting differences in the sensitivities of distinct gene signatures.

Monitoring the accumulation of fluorescently labeled phospholipids in cell cultures provides an accurate screen for drugs that induce phospholipidosis.

A large number of cationic amphiphilic drugs (CADs) are known to cause phospholipidosis (PLD) in vivo. In the present study, we have built upon our previous findings to further qualify the use of a fluorescently labeled phospholipid-based cell-culture assay to detect PLD-inducing drugs. In this paper, we demonstrate that 12 PLD-negative compounds and 11 drugs known to cause PLD in vivo are all correctly identified by using this assay. Interestingly, we found that in cells treated with certain CADs, the fluorescent phospholipid was sequestered in a very specific punctate pattern, which overlapped strongly with the staining pattern seen with a lysosomal marker protein. Our data also show that false positives can be generated with the fluorescence assay when compounds are used at concentrations that cause a >30% decrease in cell number in this assay. Confocal microscopy demonstrated that the staining pattern of fluorescent phospholipids in these cases may be differentiated from those of true positives by the fact that diffuse, rather than punctuate, fluorescence is observed. These studies confirm and expand our previous results showing that the fluorescent phospholipid assay is a highly sensitive, specific tool for detecting PLD-inducing drugs, if care is taken to rule out cytotoxicity-related artifact.

Temporal evaluation of CYP mRNA in mice administered with prototypical P450 inducers: comparison with conventional protein/enzyme methods.

Assessment of cytochrome P450 (CYP) induction at the mRNA level in preclinical rodent studies has gained interest in recent years, but there are still concerns regarding correlations between the mRNA and the enzyme activity levels, especially in mice. The purpose of the present study was to systematically evaluate patterns of temporal changes of CYPs 1a1, 1a2, 2b10, 3a11, and 4a10 at mRNA, protein, and activity levels in order to determine to what extent mRNA levels could be used either qualitatively or quantitatively for the assessment of CYP enzyme induction. In this study, livers from male CD-1 mice treated daily with beta-naphthoflavone, phenobarbital, dexamethasone, clofibrate, and control vehicles were collected for RNA and microsomal analysis after 0.5, 1, 2, 4, and 8 days of daily dose. The results revealed a good correlation among mRNA, protein, and enzyme activity levels, with the best correlation at the time points between Days 2 and 8, suggesting that the appropriate time to monitor CYP mRNA may be beyond Day 2 of chemical treatments. Based on these results, we concluded that the mRNA approach is a useful tool to monitor CYP induction in mice, particularly when treatment duration is beyond 2 days.

In vitro detection of drug-induced phospholipidosis using gene expression and fluorescent phospholipid based methodologies.

Phospholipidosis (PLD) is characterized by the excessive intracellular accumulation of phospholipids. It is well established that a large number of cationic amphiphilic drugs have the potential to induce PLD. In the present study, we describe two facile in vitro methods to determine the PLD-inducing potential of a molecule. The first approach is based on a recent study by (Sawada et al., 2005, Toxicol. Sci. 83, 282-292) in which 17 genes were identified as potential biomarkers of PLD in HepG2 cells. To confirm the utility of this gene panel, we treated HepG2 cells with PLD-positive and -negative compounds and then analyzed gene expression using real-time PCR. Our initial analysis, which used a single dose of each drug, correctly identified five of eight positive compounds and four of four negative compounds. We then increased the doses of the three false negatives (amiodarone, tamoxifen, and loratadine) and found that the changes in gene expression became large enough to correctly identify them as PLD-inducing drugs. Our results suggest that a range of concentrations should be used to increase the accuracy of prediction in this assay. Our second approach utilized a fluorescently labeled phospholipid (LipidTox) which was added to the media of growing HepG2 cells along with compounds positive and negative for PLD. Phospholipid accumulation was determined using confocal microscopy and, more quantitatively, using a 96-well plate assay and a fluorescent plate reader. Using an expanded set of compounds, we show that this assay correctly identified 100% of PLD-positive and -negative compounds. Dose-dependent increases in intracellular fluorescent phospholipid accumulation were observed. We found that this assay was less time consuming, more sensitive, and higher throughput than gene expression analysis. To our knowledge, this study represents the first validation of the use of LipidTox in identifying drugs that can induce PLD.

Assessment of atypical DNA intercalating agents in biological and in silico systems.

Non-covalent genotoxic interaction between DNA and classical planar fused-ring intercalating agents, has been well understood for some time especially in the context of frameshift mutagenesis in bacterial systems. Recent evidence, however, suggests that a rather wide structural range of small non-fused ring molecules may also be capable of partial or complete DNA intercalation in mammalian cells. The present paper will review recent studies on the identification and characterization of such atypically-structured molecules utilizing both cell-based and three-dimensional computational analyses focusing principally on prediction and detection of these atypical molecules. Mechanistic aspects of genotoxicity of such non-covalent binding molecules, with emphasis on marketed pharmaceuticals, will also be discussed. A review and presentation of new data using catalytic DNA topo II inhibitors, confirms the notion that topoisomerase II poisoning arising via intercalation is the major mechanism of genotoxicity of these drugs.

Toxicogenomics in drug discovery and development: mechanistic analysis of compound/class-dependent effects using the DrugMatrix database.

A range of genomics technologies are increasingly becoming integrated with existing scientific disciplines to broaden and strengthen existing capabilities and open new avenues of research in drug discovery and development. Examples of these new research fields are proteomics, pharmacogenomics, metabolomics and toxicogenomics. Here we review the application of toxicogenomics to improve the evaluation of drug safety, mechanism of action and toxicity in the drug discovery and development process.

DNA intercalative potential of marketed drugs testing positive in in vitro cytogenetics assays.

We have previously noted that the Physicians' Desk Reference (PDR) contains over 80 instances in which a drug elicited a positive genotoxic response in one or more in vitro assays, despite having no obvious structural features predictive of covalent drug/DNA interactive potential or known mechanistic basis. Furthermore, in most cases, these drugs were "missed" by computational genotoxicity-predicting models such as DEREK, MCASE and TOPKAT. We have previously reported the application of a V79 cell-based model and a 3D DNA docking model for predicting non-covalent chemical/DNA interactions. Those studies suggested that molecules that are very widely structurally diverse may be capable of intercalating into DNA. To determine whether such non-covalent drug/DNA interactions might be involved in unexpected drug genotoxicity, we evaluated, using both models where possible, 56 marketed pharmaceuticals, 40 of which were reported as being clastogenic in in vitro cytogenetics assays (chromosome aberrations/mouse lymphoma assay). As seen before, the two approaches showed good concordance (62%) and 26 of the 40 (65%) drugs exhibiting in vitro clastogenicity were predicted as intercalators by one or both methods. This finding provides support for the hypothesis that non-covalent DNA interaction may be a common mechanism of clastogenicity for many drugs having no obvious structural alerts for covalent DNA interaction.

SFTG international collaborative study on in vitro micronucleus test III. Using CHO cells.

In this report, results are presented from an international study of the in vitro micronucleus assay using Chinese hamster ovary cells. This study was coordinated by an organizing committee supported by the SFTG (the French branch of the European Environmental Mutagen Society). Test chemicals included mannitol, bleomycin, cytosine arabinoside, urethane and diethylstilboestrol. Mitomycin C was used as a positive control. Each chemical was evaluated in at least two laboratories following a variety of different protocols (short and long exposures, varying recovery times, with and without cytochalasin B) in order to help determine a standard protocol for routine testing in Chinese hamster ovary cells. Mannitol and urethane were negative, while bleomycin, cytosine arabinoside and diethylstilboestrol induced a dose dependent increase in micronucleated cells. In the presence of cytochalasin B, increases in micronuclei were observed in binucleated as well as mononucleated cells in cultures treated with bleomycin, cytosine arabinoside or diethylstilboestrol. Importantly, all three of these chemicals were detected in each of the different treatment/recovery regimens. No differences were seen in the sensitivity or accuracy of the responses in the presence of absence of cytochalasin B. Overall, these results demonstrate the suitability of Chinese hamster ovary cells for the in vitro micronucleus assay.

Computational prediction of genotoxicity: room for improvement.

Decades of mutagenesis and clastogenesis studies have yielded enough structure-activity-relationship (SAR) information to make feasible the construction of computational models for prediction of endpoints based on molecular structure and reactivity. Although there is cause for optimism that these approaches might someday reduce or eliminate the need for actual genotoxicity testing, we are in fact a long way from this. We provide an overview of the state of the art of such approaches, dissecting out how these models are suboptimal. It is clear that current programs still have limited predictive capabilities. We propose that one of the major contributing factors for the inherent lack of sensitivity (typically 50-60%) is inadequate coverage of non-covalent DNA interactions. Suboptimal specificity can be partly attributed to chemical space considerations with associated non-causal activity correlations.

Toward a greater appreciation of noncovalent chemical/DNA interactions: application of biological and computational approaches.

Noncovalent DNA interactions, e.g., DNA intercalation and DNA groove-binding, have not been well studied relative to covalent interactions largely due to the inability of predicting and detecting such events in intact cells. We have adapted an in vitro bleomycin amplification method for DNA intercalation for use in cultured V79 Chinese hamster cells and have validated this approach through the use of a three-dimensional DNA computational docking model that quantifies potential strength of DNA intercalative binding based on electrostatics and hydrogen bonding. For many structural classes of molecules, DNA intercalation is necessary but not sufficient for genotoxicity. The present article reviews our progress to date in predicting and confirming noncovalent binding of drugs and other chemicals and in understanding the mechanistic relationship between intercalation and genotoxicity.

Evaluation of the clastogenic, DNA intercalative, and topoisomerase II-interactive properties of bioflavonoids in Chinese hamster V79 cells.

Bioflavonoids are naturally occurring polyphenols with intriguing and varied therapeutic and chemoprotective activities generally ascribed to their antioxidant properties. However, many flavonoids have also been shown to be genotoxic in a variety of prokaryotic, eukaryotic, and in vivo systems. The mechanistic basis for this genotoxicity has not been fully elucidated, although structure-activity relationship studies have identified requisite flavonoid structural features. We utilized Chinese hamster V79 cells to evaluate the relationships between DNA intercalation ability, topoisomerase II interactions, reactive oxygen species (ROS) generation, and clastogenicity in a series of 14 bioflavonoids. Five of the flavonoids examined, luteolin, quercetin, genistein, apigenin, and acacetin, were strongly clastogenic. This clastogenicity was shown to require DNA intercalation (with the exception of genistein) and was substantially reduced by catalytic inhibitors of DNA topoisomerase II. The transition metals Cu(II) and Mn(II) formed chelates with and/or modified the structure and biological activity of some flavonoids but no consistent relationship could be demonstrated between metal reactivity and clastogenicity. There was no clear association between generation of ROS and clastogenicity. The data presented herein are consistent with a model in which the genotoxicity of most flavonoids arises via DNA intercalation and topo II poisoning, likely mediated through metabolism to flavonoid quinones. Interestingly, other flavonoids such as myricetin, daidzein, baicalein, fisetin, and galangin were catalytic topo II inhibitors, rather than poisons. These studies further validate the use of cell-based approaches for detecting drug/topo II interactions and raise interesting questions relating to biological and chemical mechanisms of flavonoids.

Evaluation of DNA intercalation potential of pharmaceuticals and other chemicals by cell-based and three-dimensional computational approaches.

To what extent noncovalent chemical-DNA interactions, in particular weak nonbonded DNA intercalation, contribute to genotoxic responses in mammalian cells has not been fully elucidated. Moreover, with the exception of predominantly flat, multiple-fused-ring structures, our ability to predict intercalation ability of novel compounds is nearly completely lacking. Computational programs such as DEREK and MCASE recognize primarily those molecules that can form irreversible covalent adducts with DNA since their learning sets, for the most part, have not been populated by compounds for which a relationship between noncovalent interaction and genotoxicity exists. We describe here a novel three-dimensional (3D) computational DNA-docking model for prediction of DNA intercalative activity of molecules with both classical and nonclassical intercalating structures. The 3D docking results show a remarkable concordance with results obtained from testing these molecules directly in the Chinese hamster V79 cell-based bleomycin amplification system suggesting that either or both of these approaches may have utility in defining noncovalent chemical-DNA interactions. The ability to predict and/or demonstrate cellular DNA intercalation of novel molecules may well provide fresh insights into the nature and mechanistic basis of structurally unexpected genotoxicity observed during safety testing.