Toxicity of dolastatin 10 in mice, rats and dogs and its clinical relevance.

PURPOSE: Dolastatin 10 (DOL 10), an oligopeptide isolated from the sea hare Dolabella auricularia, has been shown to be a highly potent cytotoxic agent in a variety of human tumor cell lines. The purpose of this study was to conduct preclinical toxicity evaluations to determine the target organ(s) of toxicity and its reversibility, the dose-limiting toxicity and the maximum tolerated dose (MTD), and to use this information for arriving at a safe starting dose and dose schedule for phase I clinical trails. METHODS: DOL10 was administered as a single intravenous bolus dose to CD2F1 mice, Fischer-344 rats and beagle dogs. Endpoints evaluated included clinical observations, body weights, hematology, serum clinical chemistry, and microscopic pathology of tissues. RESULTS: The MTD (i. e. the highest dose that did not cause lethality but produced substantial toxicity) was approximately 1350 microg/m(2) body surface area (450 microg/kg) in mice, 450 microg/m(2) (75 microg/kg) in rats and /=1350 microg/m(2) in mice, >/=150 microg/m(2) in rats and >/=400 microg/m(2) in dogs. Decreased weight gain or actual weight loss was observed at doses >/=1350 microg/m(2) in mice, >/=600 microg/m(2) in rats and >/=450 microg/m(2) in dogs. In all three species, the primary target organ of toxicity was the bone marrow, as indicated by decreases in the numbers of erythroid cells, myeloid cells, and megakaryocytes in the femoral bone marrow and by decreased white blood cell (WBC) and reticulocyte counts in peripheral blood. Marked neutropenia (i.e. >50% decrease compared to control animal or baseline values) was the principal effect on WBCs and occurred within a week of dosing. A mild anemia was evident 1 week after administering the drug to rats and dogs. The hematologic effects were transient and reversed by study termination. Other lesions at the MTD levels were cellular depletion and necrosis in lymphoid organs (rats and dogs), marked depletion of extramedullary hematopoietic cellular elements in the spleen (rats), thymic atrophy (mice and dogs), and minimal cellular necrosis in the ileum (rats). More extensive and severe pathology was observed in animals sacrificed in a moribund condition or found dead. CONCLUSIONS: Myelotoxicity was dose-limiting in all three species with mice being the least sensitive. In a phase I clinical trial, granulocytopenia was dose-limiting. Moreover, the MTD of DOL10 for rats and dogs is comparable to the human MTD. Therefore, the results from the preclinical toxicology studies correctly predicted a safe starting dose, the dose-limiting toxicity, and the MTD in humans.

A strategy for the application of transgenic rodent mutagenesis assays.

The past several years have seen an enormous increase in the development and use of transgenic animal models to measure mutations in specific inserted reporter genes. These systems provide gene mutation data in vivo in a wide range of relevant tissues. Numerous laboratories are now using these systems with consistent results. This paper describes the unique niche that transgenic mutagenesis systems can fill in product development and registration strategies. In addition to tissue-specific mechanistic studies, transgenic assays are available to follow up mutagenic effects demonstrated in Salmonella, Escherichia coli, mouse lymphoma (L5178Y) cells, or other in vitro systems.

Mutational spectrum of dimethylnitrosamine in the liver of 3- and 6-week-old lacI transgenic mice.

We determined the spectrum of mutations in the lacI gene in the liver of Big Blue(R) transgenic mice after exposure to five daily doses of 2 mg/kg dimethylnitrosamine (DMN) at 3 and 6 weeks of age. This dose has been reported to increase the mutant frequency 9-fold when the animals are 3 weeks old. The lacI mutations recovered when treated at 3 weeks consist of mainly G:C --> A:T transitions, predominantly at non-CpG sites, and thus are consistent with mutagenesis by DMN. No increase in mutant frequency was reported when the mice were treated at 6 weeks of age. As we have previously shown that changes in mutational spectrum can be detected even when no statistically significant increase in mutant frequency is seen, we also examined the spectrum after treatment at 6 weeks. No changes from the spontaneous spectrum were detected. The comparison of the outcome of DMN treatment at 3 and 6 weeks confirms a change in metabolic activation, adduct removal, or mutation fixation between 3 and 6 weeks of age.

Mutagenic response to benzene and tris(2,3-dibromopropyl)-phosphate in the lambda lacI transgenic mouse mutation assay: a standardized approach to in vivo mutation analysis.

The genotoxic response of benzene and tris(2,3-dibromopropyl)-phosphate (TDBP) have been evaluated in several tissues using the standardized lambda/lacI (Big Blue) transgenic mouse mutation assay. Separate groups of four to five male B6C3F1 transgenic lambda/lacI mice were given oral administrations of benzene or TDBP at varying concentrations. Tissues evaluated include lung, bone marrow, and spleen in benzene-treated animals, and liver, kidney, and stomach in TDBP-treated animals. Significant increases in lacI mutations were observed in the spleen and bone marrow of benzene treated mice, and the kidneys of TDBP-treated mice. Where applicable, mutagenesis patterns of tissue sensitivity were consistent with what has been observed previously in other assays. In addition, mutagenicity in tissues not traditionally evaluated for mutations correlated to sites of carcinogenicity for the chemicals tested.

Spectrum of mutations in kidney, stomach, and liver from lacI transgenic mice recovered after treatment with tris(2,3-dibromopropyl)phosphate.

The flame retardant tris(2,3-dibromopropyl)phosphate (TDBP), once used in cotton sleep wear for children, is presently banned from commerce. It produces tumors in rodents in both a sex- and tissue-specific manner. The kidney is the main target for tumor formation in male and female rats, as well as in male mice. In contrast, tumors are formed in the liver of female animals. We have used lacI transgenic male B6C3F1 mice (Big Blue) to examine the induction of mutation in kidney, liver, and stomach after exposure to 150 mg/kg (2 days), 300 mg/kg (4 days), and 600 mg/kg (4 days) of TDBP. At the highest dose, the mutant frequency was approximately 50% above control values in the kidney (P < 0.01). A smaller increase was observed in the liver (P = 0.07), while no increase was seen in the stomach (P = 0.28). Sequence analysis of the recovered mutants showed a TDBP-specific change in mutation spectrum in kidney, which was not observed in liver and stomach. In kidney, a dose-dependent decrease in G:C-->A:T transitions, including at 5'-CpG-3' sites, was observed. This was accompanied by an increase in the loss of single G:C base pairs from approximately 3% to 15%. These results illustrate both the sensitivity and specificity of the lacI transgenic system in the analysis of tissue-specific mutation. This study also reinforces the importance of examining mutational spectra when mutant induction levels are low.

Evaluation of the potential of riddelliine to induce unscheduled DNA synthesis, S-phase synthesis, or micronuclei following in vivo treatment with multiple doses.

Riddelliine (RID) is a pyrrolizidine alkaloid found in plants of the genera Crotalaria, Amsinckia, and Senecio in the United States. RID has been extensively studied in a wide variety of in vitro short-term genotoxicity tests and has yielded positive responses in most test systems; however, there are fewer data available on the effects of RID in in vivo assays and no data under repeat-dose regimens. We have evaluated the ability of RID to induce unscheduled DNA synthesis (UDS) in hepatocytes, S-phase synthesis (SPS) in hepatocytes, and micronuclei in bone marrow from animals dosed for 5 or 30 days in conjunction with prechronic toxicity testing conducted for the National Toxicology Program. Results of this study indicate that RID did not induce an increase in micronucleated polychromatic erythrocytes (PCE) in bone marrow of mice after 5 days of dosing or in PCE from rats or mice after 30 days of dosing. RID did not induce an increase in UDS in rat hepatocytes after 5 or 30 days of dosing, but it did induce an equivocal UDS response in male mice after both time points and a positive response in female mice after 30 days of dosing. RID induced significant elevations in SPS in rat hepatocytes after both 5 and 30 days of dosing, even at low doses. An increase in SPS was observed in male and female mouse hepatocytes, but only after 30 days of dosing. Rats and mice of both sexes showed a depression in SPS at higher doses. This effect may be a result of toxicity, which compromises the ability of the liver to regenerate. These results demonstrate that repeated administration of test chemicals may alter the genotoxic response to chemicals.

Measurement of unscheduled DNA synthesis and S-phase synthesis in rodent hepatocytes following in vivo treatment: testing of 24 compounds.

The in vivo-in vitro hepatocyte DNA repair assay has been shown to be useful for studying genotoxic hepatocarcinogens. In addition, measurement of S-phase synthesis (SPS) provides an indirect indicator of hepatocellular proliferation, which may be an important mechanism in rodent carcinogenesis. This assay was used to examine 24 chemicals for their ability to induce unscheduled DNA synthesis (UDS) or SPS in Fischer-344 rats or B6C3F1 mice following in vivo treatment. Hepatocytes were isolated by liver perfusion and incubated with 3H-thymidine following in vivo treatment by gavage. UDS was measured by quantitative autoradiography as net grains/nucleus (NG). Controls from both sexes of both species yielded less than 0.0 NG. Chemicals chosen for testing were from the National Toxicology Program (NTP) genetic toxicology testing program and most were also evaluated in long-term animal studies conducted by the NTP. 11-Aminoundecanoic acid, benzyl acetate, bis(2-chloro-1-methylethyl)ether (BCMEE), C.I. Solvent Yellow 14, cinnamaldehyde, cinnamyl anthranilate, dichloromethane, dichlorvos, glutaraldehyde, 4,4'-methylenedianiline (MDA), 4-nitrotoluene, 4,4'-oxydianiline, a polybrominated biphenyl mixture (PBB), reserpine, 1,1,2,2-tetrachloroethane, 1,1,2-trichloroethane, trichloroethylene, and 2,6-xylidine all failed to induce UDS in rats and/or mice. Dinitrotoluene and Michler's Ketone induced positive UDS response in rat, while N-nitrosodiethanolamine and selenium sulfide induced equivocal UDS results in mouse and rat, respectively. BCMEE, bromoform, chloroform, PBB, 1,1,2-trichloroethane, and trichloroethylene were all potent inducers of SPS in mouse liver, while C.I. Solvent Yellow 14, and 1,1,2,2-tetrachloroethane yielded equivocal SPS results in rat and mouse, respectively. These results indicate that most of the test compounds do not induce UDS in the liver; however, the significant S-phase responses induced by many of these compounds, especially the halogenated solvents, may be an important mechanism in their hepatocarcinogenicity.

Comparative studies on the genotoxic activity of mainstream smoke condensate from cigarettes which burn or only heat tobacco.

The in vitro genotoxic activity of mainstream cigarette smoke condensate (CSC) from cigarettes which heat but do not burn tobacco was compared to that of CSC from cigarettes which burn tobacco. CSCs from five cigarettes were compared. Three of the cigarettes [the Kentucky reference research cigarette (1R4F), a commercially available ultra-low tar brand (ULT) and a commercially available ultra-low tar menthol brand (ULT-menthol]) burn tobacco while two of the cigarettes [a regular (TEST) and a menthol (TEST-menthol]) heat tobacco. CSC from all cigarettes were collected by identical standard techniques, which involved collecting mainstream smoke particulate matter on Cambridge filter pads under FTC smoking conditions. The pads were extracted with DMSO, and the CSCs obtained [10 mg total particulate matter (TPM)/ml DMSO] were evaluated at identical concentrations in an in vitro genetic toxicology test battery. CSCs from 1R4F, ULT, and ULT-menthol cigarettes were mutagenic in Ames bacterial strains TA98, TA100, TA1537, and TA1538 in the presence of metabolic activation (S9 from Aroclor-induced rat liver) but negative in strain TA1535. In the absence of metabolic activation, 1R4F, ULT, and ULT-menthol CSCs were not mutagenic except for a weak response in strain TA1537 for the 1R4F and ULT CSCs. TEST and TEST-menthol CSCs were nonmutagenic in all five bacterial strains, both with and without metabolic activation. CSCs from 1R4F, ULT, and ULT-menthol cigarettes were positive in the CHO-chromosomal aberration assay and in the CHO--sister chromatid exchange assay both with and without metabolic activation while TEST and TEST-menthol CSCs were negative in both assays, either with or without metabolic activation. CSCs from 1R4F, ULT, and ULT-menthol cigarettes were weakly positive in inducing DNA repair in cultured rat hepatocytes while TEST and TEST-menthol CSCs were negative in this assay. All five CSCs were nonmutagenic in the CHO-HGPRT assay both with and without metabolic activation. CSCs from the 1R4F, ULT, and ULT-menthol cigarettes were cytotoxic in the CHO-HGPRT assay, both with and without metabolic activation, while TEST and TEST-menthol CSCs were not cytotoxic under either condition. These results demonstrate that mainstream CSCs from the TEST and TEST-menthol cigarettes are neither genotoxic nor cytotoxic under conditions where CSCs from 1R4F, ULT, and ULT-menthol cigarettes are genotoxic and/or cytotoxic in a concentration-dependent manner.

Genetic toxicology testing of the antimalarial drugs chloroquine and a new analog, AQ-13.

AQ-13 ([N1-(7-chloro-quinolin-4yl)-3-(N3,N3-diethylamino)propylamine] dihydrochloride trihydrate) is an aminoquinoline antimalarial drug that is effective against chloroquine-resistant strains of Plasmodium falciparum. It is structurally similar to the widely used chloroquine diphosphate (CQ). We evaluated these drugs in the three assays currently recommended by the International Conference on Harmonization (ICH): bacterial mutagenesis in Salmonella typhimurium and Escherichia coli, mammalian cell mutagenesis in L5178Y mouse lymphoma cells, and micronucleus induction in rat bone marrow. A small but statistically significant increase in revertant colonies was produced by CQ with Salmonella tester strain TA98 without metabolic activation (MA) and by AQ-13 with strain TA1537 both with and without MA. In L5178Y cells, testing of CQ and AQ-13 up to cytotoxic concentrations with and without MA produced no increase in mutant colonies and no increase in the numbers of small colonies. Slight decreases in the ratio of polychromatic erythrocytes (PCE) to red blood cells (RBC) were observed in male and female rats treated with CQ and in females only treated with AQ-13; however, none of these changes was statistically significant. No increases in the frequency of micronucleated PCE were observed at any dose level of CQ or AQ-13. Although both CQ and AQ-13 showed weak bacterial mutagenicity, this mutagenic effect was not confirmed in either the mouse lymphoma mutagenesis assay or the micronucleus assay. These results indicate that CQ and AQ-13 should pose minimal risk of genotoxic damage in human populations being administered these drugs.

Evaluation of positive controls for the in vitro unscheduled DNA synthesis assay using hepatocytes from induced (Aroclor 1254) and uninduced male cynomolgus monkey.

We have evaluated the use of four different positive control compounds for assessing UDS in monkey hepatocytes and have found three of these, methylmethanesulfonate, benzo[a]pyrene, and dimethylbenz[a]anthracene, to produce strong positive responses in vitro. Dimethylnitrosamine induced only weak responses. We also report that the strength of the response induced by procarcinogens was not enhanced in hepatocytes taken from Aroclor 1254-pretreated monkeys, even though substantial induction of cytochrome P450 enzymes was demonstrated in these cells. These studies raise the question of the utility of employing an in vivo induction system to enhance the monkey UDS assay.

Micronucleated erythrocyte frequency in peripheral blood of B6C3F(1) mice from short-term, prechronic, and chronic studies of the NTP carcinogenesis bioassay program.

The mouse peripheral blood micronucleus (MN) test was performed on samples collected from 20 short-term, 67 subchronic, and 5 chronic toxicity and carcinogenicity studies conducted by the National Toxicology Program (NTP). Data are presented for studies not previously published. Aspects of protocol that distinguish this test from conventional short-term bone marrow MN tests are duration of exposure, and absence of repeat tests and concurrent positive controls. Furthermore, in contrast to short-term bone marrow MN tests where scoring is limited to polychromatic erythrocytes (PCE), longer term studies using peripheral blood may evaluate MN in both, or either, the normochromatic (NCE) or PCE populations. The incidence of MN-PCE provides an index of damage induced within 72 hr of sampling, whereas the incidence of MN in the NCE population at steady state provides an index of average damage during the 30-day period preceding sampling. The mouse peripheral blood MN test has been proposed as a useful adjunct to rodent toxicity tests and has been effectively incorporated as a routine part of overall toxicity testing by the NTP. Data derived from peripheral blood MN analyses of dosed animals provide a useful indication of the in vivo potential for induced genetic damage and supply an important piece of evidence to be considered in the overall assessment of toxicity and health risk of a particular chemical. Although results indicate that the test has low sensitivity for prediction of carcinogenicity, a convincingly positive result in this assay appears to be highly predictive of rodent carcinogenicity.

In vivo transgenic mutation assays.

Transgenic rodent gene mutation models provide quick and statistically reliable assays for mutations in the DNA from any tissue. For regulatory applications, assays should be based on neutral genes, be generally available in several laboratories, and be readily transferable. Five or fewer repeated treatments are inadequate to conclude that a compound is negative but more than 90 daily treatments may risk complications. A sampling time of 35 days is suitable for most tissues and chemicals, while shorter sampling times might be appropriate for highly proliferative tissues. For phage-based assays, 5 to 10 animals per group should be analyzed, assuming a spontaneous mutant frequency (MF) of approximately 3 x 10(-5) mutants/locus and 125,000-300,000 plaque or colony forming units (PFU or CFU) per tissue. Data should be generated for two dose groups but three should be treated, at the maximum tolerated dose (MTD), two-thirds the MTD, and one-third the MTD. Concurrent positive control animals are only necessary during validation, but positive control DNA must be included in each plating. Tissues should be processed and analyzed in a block design and the total number of PFUs or CFUs and the MF for each tissue and animal reported. Sequencing data would not normally be required but might provide useful additional information in specific circumstances. Statistical tests used should consider the animal as the experimental unit. Nonparametric statistical tests are recommended. A positive result is a statistically significant dose-response and/or statistically significant increase in any dose group compared to concurrent negative controls using an appropriate statistical model. A negative result is statistically nonsignificant with all mean MF within two standard deviations of the control.

Professional toxicology societies: web based resources.

The number of digital tools available for use by researchers continues to rapidly increase as does the quantity of information available on the web. However, judging the reliability, quality and timeliness of this information is not always easy. One source of information about the profession of toxicology comes from the web sites of the various professional organizations that exist in this field. Given the broad nature of toxicology, there are a large number of such organizations. Some serve broader segments of the profession, while others have a more narrow focus. The web sites they have created are similarly variable, although there are some consistencies in the type of information that is provided. These include mission statements, information on joining the organization, and details on their annual meetings. There is also a consistent decision by these sites not to provide toxicologic information per se, although links to sites that do contain such information are occasionally provided. Overall, the information and tools provided by professional toxicology societies around the world appears to be similar in both quantity and quality to other scientific organizations. It is also apparent that this information is rapidly evolving and will likely be much different within the next 5 years.

Transgenic models for detection of mutations in tumors and normal tissues of rodents.

Transgenic rodents that contain easily retrievable target genes allow the rapid quantitation of mutations in any tissue from which DNA can be isolated. We are using the Stratagene Big Blue transgenic mouse system that contains a lacI target and an alpha lacZ reporter gene to study the parameters that affect mutations. We have evaluated a number of chemicals to determine mutant frequency (MF) in specific target tissues of C57Bl/6 and B6C3F1 mice. The correlation between mutagenesis and carcinogenesis in this system is excellent. For example, the liver carcinogen dimethylnitrosamine produces significant increases in MF in mouse liver, whereas the nonhepatocarcinogenic mutagen methylmethane sulfonate does not. We have also evaluated the induction of mutations by radiation and demonstrated that this system is suitable for the study of agents that produce deletion mutations. This system is also useful for studying changes in MF in developing tumors. We have used an initiation-promotion protocol to induce hepatocellular carcinomas, and we then measured MF in normal liver, tumors, and metastases from these mice. Animals initiated with diethylnitrosamine maintain an elevated MF in normal liver, even 1 year after initiation. This MF increases exponentially in developing liver tumors, possibly owing to a breakdown in the fidelity of DNA replication and DNA repair in tumors. This system offers a unique tool for the study of mutations induced in specific target tissues of rodents and should become an important assay for evaluating the mutagenic risk of drugs and chemicals.

Genotoxicity, toxicity, and carcinogenicity of the antihistamine methapyrilene.

The antihistamine methapyrilene hydrochloride (MP) has been shown to be a potent hepatocarcinogen in rats, but not in hamsters or guinea pigs. This finding is in contrast to the relative nongenotoxicity of this compound. MP has been evaluated for genotoxicity in a wide variety of short-term tests and has generally demonstrated little genotoxic activity. One exception to this is the mouse lymphoma L5178Y mutagenesis assay, in which MP produced a very significant increase in small colony mutants with concomitant chromosomal damage in these cells. MP also induced positive responses in several cell transformation assays. One potentially very significant effect of MP is that it induces a large increase in hepatic cell proliferation coupled with mitochondrial proliferation in the livers of treated animals. This effect is discussed as a possible mechanism of liver tumor induction in rats.

Factors that affect the sensitivity of the in vivo-in vitro hepatocyte DNA repair assay in the male rat.

Measurement of chemically induced DNA repair as unscheduled DNA synthesis (UDS) in rat hepatocytes following in vivo exposure has been shown to be a useful indicator of the genotoxicity of chemicals in rat liver. We have examined some of the parameters of this assay in an attempt to increase its sensitivity and reduce cytoplasmic backgrounds. Fischer-344 rats were treated with a low dose of a known positive chemical, water, or corn oil. Livers were perfused with a collagenase solution and isolated hepatocytes were incubated with [3H]thymidine (3H-TdR) followed by overnight incubation in unlabeled TdR, then cell fixation and washing. UDS was measured by quantitative autoradiographic grain-counting as net grains/nucleus (NG). Incubation in 3H-TdR ranging in age from 1 week to more than 12 months gave highly variable background (BKG) and NG counts and a slight overall decrease in NG when the 3H-TdR used was more than 4 months old. Control BKG was 3 times higher after 19 h than after 4 h of incubation in 3H-TdR, with little change in NG. Incubation in unlabeled TdR also reduced BKG significantly. Reduction of autoradiogram exposure from two to one week cut BKG in half without significantly reducing NG. A half-hour wash in fixative (1:3 acetic acid:ethanol) followed by two water washes was as effective in reducing BKG as three 10-min washes in fixative followed by 6 water washes, and resulted in better overall cell attachment. An examination of the distribution of historical control data shows that vehicle control animals never exceed zero NG. This suggests that any NG response greater than zero should be viewed as a possible positive response.

Analysis of the mutagenic potential of ENU and MMS in germ cells of male C57BL/6 lacI transgenic mice.

Mutant frequencies in male germ cells were determined in mice 3 days after exposure to saline, methylmethane sulfonate (MMS), or ethylnitrosourea (ENU). DNA was isolated from seminiferous tubules by a modified version of the drop dialysis method. A 5-fold increase in mutant frequency was observed in mice treated with ENU. No statistically significant increase was observed in mice treated with MMS.

The in vitro unscheduled DNA synthesis (UDS) assay in rat primary hepatocytes: evaluation of 24 drug candidates.

The in vitro unscheduled DNA synthesis assay (UDS) is part of the routine genetic toxicology screening at The Upjohn Company. The purpose of this paper is to report results for 24 drug candidates which were tested as coded compounds. These compounds are very diverse in chemical structure and represent classes of compounds selected because of biological activity in a variety of preliminary drug efficacy screens. None of the compounds reported here produced an increase in UDS, and therefore, the UDS results with these materials do not suggest potential for mutagenesis or carcinogenesis.

A protocol and guide for the in vitro rat hepatocyte DNA-repair assay.

The in vitro rat-hepatocyte DNA-repair assay is a valuable tool in assessing the genotoxic activity of chemical agents. An advantage of the assay is that the target cells themselves are metabolically competent, so that the patterns of metabolic activation and detoxification closely reflect those in the whole animal. This article provides a typical procedure and guidelines for conducting the rat in vitro hepatocyte DNA-repair assay.

A protocol and guide for the in vivo rat hepatocyte DNA-repair assay.

The in vivo rat hepatocyte DNA-repair assay is a valuable tool in assessing the genotoxic activity of chemical agents. An advantage of the system is that it reflects the complex patterns of uptake, distribution, metabolism, detoxification and excretion that actually occur in the whole animal. This article provides a typical procedure and guidelines for conducting the rat in vivo hepatocyte DNA-repair assay.