Neuroprotective effect of 3,5-di-O-caffeoylquinic acid on SH-SY5Y cells and senescence-accelerated-prone mice 8 through the up-regulation of phosphoglycerate kinase-1.

As aged population dramatically increases in these decades, efforts should be made on the intervention for curing age-associated neurologic degenerative diseases such as Alzheimer's disease (AD). Caffeoylquinic acid (CQA), an antioxidant component and its derivatives are natural functional compounds isolated from a variety of plants. In this study, we determined the neuroprotective effect of 3,5-di-O-CQA on Abeta(1-42) treated SH-SY5Y cells using MTT assay. To investigate the possible neuroprotective mechanism of 3,5-di-O-CQA, we performed proteomics analysis, real-time PCR analysis and measurement of the intracellular ATP level. In addition, we carried out the measurement of escape latency time to find the hidden platform in Morris water maze (MWM), real-time PCR using senescence-accelerated-prone mice (SAMP) 8 and senescence-accelerated-resistant mice (SAMR) 1 mice. Results showed that 3,5-di-O-CQA had neuroprotective effect on Abeta (1-42) treated cells. The mRNA expression of glycolytic enzyme (phosphoglycerate kinase-1; PGK1) and intracellular ATP level were increased in 3,5-di-O-CQA treated SH-SY5Y cells. We also found that 3,5-di-O-CQA administration induced the improvement of spatial learning and memory on SAMP8 mice, and the overexpression of PGK1 mRNA. These findings suggest that 3,5-di-O-CQA has a neuroprotective effect on neuron through the upregulation of PGK1 expression and ATP production activation.

The alkaline single cell electrophoresis assay with eight mouse organs: results with 22 mono-functional alkylating agents (including 9 dialkyl N-nitrosoamines) and 10 DNA crosslinkers.

The genotoxicity of 22 mono-functional alkylating agents (including 9 dialkyl N-nitrosoamines) and 10 DNA crosslinkers selected from IARC (International Agency for Research on Cancer) groups 1, 2A, and 2B was evaluated in eight mouse organs with the alkaline single cell gel electrophoresis (SCGE) (comet) assay. Groups of four mice were treated once intraperitoneally at the dose at which micronucleus tests had been conducted, and the stomach, colon, liver, kidney, bladder, lung, brain, and bone marrow were sampled 3, 8, and/or 24 h later. All chemicals were positive in the SCGE assay in at least one organ. Of the 22 mono-functional alkylating agents, over 50% were positive in all organs except the brain and bone marrow. The two subsets of mono-functional alkylating agents differed in their bone marrow genotoxicity: only 1 of the 9 dialkyl N-nitrosoamines was positive in bone marrow as opposed to 8 of the 13 other alkylating agents, reflecting the fact that dialkyl N-nitrosoamines are poor micronucleus inducers in hematopoietic cells. The two groups of mono-functional alkylating agents also differ in hepatic carcinogenicity in spite of the fact that they are similar in hepatic genotoxicity. While dialkyl N-nitrosoamines produce tumors primarily in mouse liver, only one (styrene-7,8-oxide) out of 10 of the other type of mono-functional alkylating agents is a mouse hepatic carcinogen. Taking into consideration our previous results showing high concordance between hepatic genotoxicity and carcinogenicity for aromatic amines and azo compounds, a possible explanation for the discrepancy might be that chemicals that require metabolic activation show high concordance between genotoxicity and carcinogenicity in the liver. A high percent of the 10 DNA crosslinkers were positive in the SCGE assay in the gastrointestinal mucosa, but less than 50% were positive in the liver and lung. In this study, we allowed 10 min alkali-unwinding to obtain low and stable control values. Considering that DNA crosslinking lesions can be detected as lowering of not only positive but also negative control values, low control values by short alkali-treatment might make it difficult to detect DNA crosslinking lesions. In conclusion, although both mono-functional alkylating agents and DNA crosslinkers are genotoxic in mouse multiple organs, the genotoxicity of DNA crosslinkers can be detected in the gastrointestinal organs even though they were given intraperitoneally followed by the short alkali-treatment.

Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing.

Atthe International Workshop on Genotoxicity Test Procedures (IWGTP) held in Washington, DC, March 25-26, 1999, an expert panel met to develop guidelines for the use of the single-cell gel (SCG)/Comet assay in genetic toxicology. The expert panel reached a consensus that the optimal version of the Comet assay for identifying agents with genotoxic activity was the alkaline (pH > 13) version of the assay developed by Singh et al. [1988]. The pH > 13 version is capable of detecting DNA single-strand breaks (SSB), alkali-labile sites (ALS), DNA-DNA/DNA-protein cross-linking, and SSB associated with incomplete excision repair sites. Relative to other genotoxicity tests, the advantages of the SCG assay include its demonstrated sensitivity for detecting low levels of DNA damage, the requirement for small numbers of cells per sample, its flexibility, its low costs, its ease of application, and the short time needed to complete a study. The expert panel decided that no single version of the alkaline (pH > 13) Comet assay was clearly superior. However, critical technical steps within the assay were discussed and guidelines developed for preparing slides with agarose gels, lysing cells to liberate DNA, exposing the liberated DNA to alkali to produce single-stranded DNA and to express ALS as SSB, electrophoresing the DNA using pH > 13 alkaline conditions, alkali neutralization, DNA staining, comet visualization, and data collection. Based on the current state of knowledge, the expert panel developed guidelines for conducting in vitro or in vivo Comet assays. The goal of the expert panel was to identify minimal standards for obtaining reproducible and reliable Comet data deemed suitable for regulatory submission. The expert panel used the current Organization for Economic Co-operation and Development (OECD) guidelines for in vitro and in vivo genetic toxicological studies as guides during the development of the corresponding in vitro and in vivo SCG assay guidelines. Guideline topics considered included initial considerations, principles of the test method, description of the test method, procedure, results, data analysis and reporting. Special consideration was given by the expert panel to the potential adverse effect of DNA degradation associated with cytotoxicity on the interpretation of Comet assay results. The expert panel also discussed related SCG methodologies that might be useful in the interpretation of positive Comet data. The related methodologies discussed included: (1) the use of different pH conditions during electrophoreses to discriminate between DNA strand breaks and ALS; (2) the use of repair enzymes or antibodies to detect specific classes of DNA damage; (3) the use of a neutral diffusion assay to identify apoptotic/necrotic cells; and (4) the use of the acellular SCG assay to evaluate the ability of a test substance to interact directly with DNA. The alkaline (pH > 13) Comet assay guidelines developed by the expert panel represent a work in progress. Additional information is needed before the assay can be critically evaluated for its utility in genetic toxicology. The information needed includes comprehensive data on the different sources of variability (e.g., cell to cell, gel to gel, run to run, culture to culture, animal to animal, experiment to experiment) intrinsic to the alkaline (pH > 3) SCG assay, the generation of a large database based on in vitro and in vivo testing using these guidelines, and the results of appropriately designed multilaboratory international validation studies.

Casein kinase II is responsible for phosphorylation of NF-L at Ser-473.

Ser-473 is solely phosphorylated in vivo in the tail region of neurofilament L (NF-L). With peptides including the native phosphorylation site, it was not possible to locate responsible kinases. We therefore adopted full-length dephosphorylated NF-L as the substrate, and employed MALDI/TOF (matrix-assisted laser desorption and ionization/time of flight) mass spectrometry and a site-specific phosphorylation-dependent antibody recognizing Ser-473 phosphorylation. The antibody showed that casein kinase I (CK I) as well as casein kinase II (CK II) phosphorylated Ser-473 in vitro, while neither GSK-3beta nor calcium/calmodulin-dependent protein kinase II did so. However, the mass spectra of the tail fragments of the phosphorylated NF-L indicated that CK II was the kinase mediating Ser-473 phosphorylation in vitro as opposed to CK I, because CK I phosphorylated another site as well as Ser-473 in vitro. The antibody also demonstrated that NF-L phosphorylated at Ser-473 was abundant in the neuronal perikarya of the rat cortex, indicating that phosphorylation of Ser-473 may take place there. This result may support the suggestion that CK II is the kinase responsible for Ser-473 phosphorylation. Despite many reports showing that CK I mediates phosphorylation of neurofilaments, CK II may phosphorylate NF-L in vivo.

Pulse exposure of cultured rat neurons to aluminum-maltol affected the axonal transport system.

Although chronic aluminum neurotoxicity has been well established, the mechanism of the toxicity has not been elucidated yet. In order to simplify the study of the aluminum neurotoxicity, we employed the pulse exposure of cultured rat cortical neurons to 250 microM aluminum-maltol for 1 h at the early stage (6 h after plating), which resulted in abnormal distribution of neurofilament L (NFL) and fast axonal transported proteins, whereas the axonal transport of tubulin, actin, and clathrin were not impaired. Otherwise, the pulse exposure of neurons at the late stage (4 days after plating) to the same concentration of aluminum-maltol did not affect the cell morphology and the distribution of NFL. The pulse exposure of cultured neurons to aluminum-maltol at the early stage might affect the axonal transport system of NFL and fast axonal transported proteins.

Evaluation of a tissue homogenization technique that isolates nuclei for the in vivo single cell gel electrophoresis (comet) assay: a collaborative study by five laboratories.

We evaluated a tissue homogenization technique that isolates nuclei for use in the in vivo comet assay. Five laboratories independently tested the technique using the liver, kidney, lung, spleen, and bone marrow of untreated and mutagen-treated male CD-1 mice. The direct mutagen methylmethanesulfonate (MMS) or the promutagen diethylnitrosamine (DEN) were injected intraperitoneally at maximum tolerated doses. Three and twenty-four hours later, the organs were removed and, except for bone marrow, were minced and homogenized and a nuclear suspension was prepared. The nuclear suspensions and bone marrow cells were used in the comet assay. None of the nuclear suspensions from the non-treated mice induced a positive response. All nuclear suspensions derived from the MMS-treated mice and those of the liver, kidney, and lung from DEN-treated mice induced positive responses in all the laboratories similarly. Reproducibility was demonstrated by five replicate studies in one laboratory. Furthermore, the organ-specific responses to MMS and DEN reflected the characteristic genotoxicity of the chemicals. We concluded from these results that the homogenization technique is a valid one to be used for mouse organs in the in vivo comet assay.

Evaluation of the rat micronucleus test with bone marrow and peripheral blood: summary of the 9th collaborative study by CSGMT/JEMS. MMS. Collaborative Study Group for the Micronucleus Test. Environmental Mutagen Society of Japan. Mammalian Mutagenicity Study Group.

The mouse has traditionally been used for the micronucleus test, with bone marrow the usual target organ. The aim of the 9th collaborative study by CSGMT was to evaluate the suitability of the rat for the micronucleus test, with bone marrow and peripheral blood as the target organ. Since the rat spleen eliminates circulating micronucleated erythrocytes, a rat peripheral blood micronucleus assay might not be feasible. Thirty-four Japanese laboratories and six overseas laboratories participated in this collaboration, and 40 chemicals were studied. As a rule, rat bone marrow and peripheral blood were analyzed using acridine orange staining. Among 36 mouse micronucleus-positive rat carcinogens, 34 of which had been evaluated by CSGMT, we observed 33 positive and three negative results with rat bone marrow and 30 positive, three equivocal, and three negative responses with rat peripheral blood. Of the two mouse micronucleus-negative rat carcinogens, acrylonitrile was positive in rat bone marrow and 4,4'-methylene bis(2-chloroaniline) was negative in both rat bone marrow and peripheral blood. Two chemicals reported to be mouse micronucleus-negative and rat-positive, azobenzene and Solvent Yellow 14, and one chemical reported to be mouse-positive and rat-negative, 1,2-dimethylhydrazine, gave positive responses in rat bone marrow and peripheral blood. The concordance between bone marrow and peripheral blood with rats was 92%. The concordance between rat and mouse erythrocytes was 88%. We concluded that the rat micronucleus assay, using either bone marrow or peripheral blood, can be used as an alternative to the mouse micronucleus assay.

Detection of DNA lesions induced by chemical mutagens by the single cell electrophoresis (Comet) assay. 1. Relationship between the onset of DNA damage and the characteristics of mutagens.

We evaluated the relationship between the onset of DNA damage and the characteristics of 5 model chemical mutagens with the single-cell gel electrophoresis (SCG) assay using L5178Y mouse lymphoma cells. We treated the cells with each chemical for 3 h and sampled them 0.21, and 45 h after treatment. DNA damage induced by UV mimetic mutagens MMS and MNU, and X-ray mimetic mutagen BLM was observed just after treatment, crosslinking agent MMC-induced DNA damage was detected 21 h after treatment, and 6-MP as an inhibitor of DNA synthesis did not induce DNA damage at any sampling time. These results suggest that the SCG assay detects DNA lesions just after treatment with UV and X-ray mimetic mutagens, but needs a waiting period after treatment with crosslinking agents.

Altered adhesion efficiency and fibronectin content in fibroblasts from schizophrenic patients.

Cultured fibroblasts from the cutaneous tissue of 16 schizophrenic patients were compared with 16 control cultured fibroblasts from the healthy subjects. The fibroblasts from the schizophrenic patients showed a decreased adhesion efficiency within 30 min after plating compared to that of the control subjects. However, after 90 min, there was no significant difference between the groups, where more than 90% of the cells from both groups had adhesed to the plate. By immunohistochemistry and western blotting using the antibodies against integrin (VLA5), talin, vinculin, fodrin, vimentin, ankyrin, plectin, fibronectin, and focal adhesion kinase (FAK), there was no significant difference in localization and amount between the groups. The amount of fibronectin released into the medium in which the fibroblast had already kept confluency showed no significant difference between the groups. However, the fibronectin content in cell lysate within 48 h after plating was significantly lower in the schizophrenic group.

Increased tau protein level in postmortem cerebrospinal fluid.

Microtubule-associated protein tau has been reported to be significantly increased in cerebrospinal fluid (CSF) of the patients with Alzheimer's disease (AD), which suggests that it is possibly a biological marker for the diagnosis of AD. The underlying mechanism of the increased tau level in CSF, however, is not known. In this study, the tau levels were compared between antemortem and postmortem CSF. The postmortem tau levels in CSF were significantly increased in all groups including AD, neurological control, and nondemented control. A striking elevation of CSF tau was observed during the postmortem change with the nondemented subjects. These findings may offer some insight into the understanding of the mechanism of the increased tau level in CSF with AD and other related disorders.

Decisive problems of zone-circulating flow injection analysis and its solution.

Although experiment and computer analysis of zone-circulating flow injection analysis (ZCFIA) data have been investigated, there are still some essential problems inherent to ZCFIA. Computer program of high dimensional modified simplex method was used for resolving peaks of ZCFIA damped response curves. Peaks are resolved on the basis of the criterion that each area of the peak surrounded by the curve and the abscissa is equal, because each sample zone circulates repeatedly in the manifold in equal volume. As a result, the peaks of the damped response curve have been resolved into each component and the curve obtained by summing these components has been proved to be equal to the original response curve. By following up the data analysis of ZCFIA, it was found that there were many conflicts in the manual analysis of data by Li. At least, the dispersion in a flow system should not be investigated by ZCFIA, and it might be studied by the single-line manifold of FIA.

Detection of DNA lesions induced by chemical mutagens by the single cell gel electrophoresis (Comet) assay. 1. Relationship between the onset of DNA damage and the characteristics of mutagens.

We evaluated the relationship between the onset of DNA damage and the characteristics of 5 model chemical mutagens with the single-cell gel electrophoresis (SCG) assay using L5178Y mouse lymphoma cells. We treated the cells with each chemical for 3 h and sampled them 0, 21, and 45 h after treatment. DNA damage induced by UV mimetic mutagens MMS and MNU, and X-ray mimetic mutagen BLM was observed just after treatment, crosslinking agent MMC-induced DNA damage was detected 21 h after treatment, and 6-MP as an inhibitor of DNA synthesis did not induce DNA damage at any sampling time. These results suggest that the SCG assay detects DNA lesions just after treatment with UV and X-ray mimetic mutagens, but needs a waiting period after treatment with crosslinking agents.

Detection of DNA lesions induced by chemical mutagens using the single-cell gel electrophoresis (comet) assay. 2. Relationship between DNA migration and alkaline condition.

The alkaline condition is an important factor for the alkaline single-cell gel electrophoresis (SCG) assay to detect the genotoxic effects of chemicals. In order to understand the relationship between DNA migration and alkaline condition, the effect of 13 model chemical mutagens with different modes of action was evaluated with the alkaline SCG assay under two different alkaline conditions (pH 12.1 and 12.6). CHO cells were sampled just after treatment for 1 h. The X-ray mimetic mutagen BLM increased DNA migration at pH 12.1 and 12.6 and the results were the same at both pH values. Six alkylating mutagens MNU, ENU, MNNG, ENNG, MMS, and EMS and one base adduct inducer 4-NQO induced a dose-dependent response only at pH 12.6. Two DNA crosslinking agents, MMC and DDP, and AMD had negative results. MMC and DDP, however, reduced the positive response of BLM, suggesting that DNA crosslinks could be detected. These results demonstrated that the alkaline condition was important factor for the alkaline SCG assay to detect the genotoxic effects of chemicals.

Temporal and regional profiles of cytoskeletal protein accumulation in the rat brain following traumatic brain injury.

To characterize the cytoskeletal aberration due to traumatic injury, temporal and regional profiles of changes in immunoreactivity of microtubule-associated protein 2 (MAP2), neurofilament heavy subunit protein (NFH) and heat shock protein 72 (HSP72) were investigated after different magnitudes of traumatic brain injury by fluid percussion. The experimental rat brain was perfusion-fixed at 1, 6 and 24 hours after traumatic brain injury. Conventional histological staining has demonstrated that the mildest traumatic brain injury (1.0 atm) induced no neuronal loss at the impact site and that neuron loss was apparent when traumatic brain injury was increased to 4.3 atm. The mildest traumatic brain injury, however, caused a significant increase in HSP72 immunoreactivity in the superficial cortical layers at the impact site as early as 1 hour after the injury. In the case of severe traumatic brain injury (4.3 atm), neuron loss was apparent in the area at the impact site, but the increase in HSP72 immunoreactivity was moderate, and it was observed only after 6 hours in the deep cortical layers under the necrotic area. The increased immunostaining of MAP2 was demonstrated in damaged axons and neuronal perikarya in the wider area surrounding the impact site at 6 and 24 hours after the injury. Six and 24 hours after the injury, perikaryal accumulation of neurofilament was observed, and the accumulated neurofilament was mostly phosphorylated. These results indicate that the severe traumatic brain injury of 4.3 atm triggers the abnormal accumulation of cytoskeletal proteins in neuronal perikarya, most probably due to an impairment of axonal transport. It is implied that the increased expression of HSP72 may be involved in the protective process of neurons after traumatic brain injury.

Classification system of complications in neuroleptic malignant syndrome.

Our group treated 13 cases of neuroleptic malignant syndrome (NMS) over a period of 8 years. Based on the clinical severity of complications, the cases were classified into three types: mild, with no complications; moderate, with only respiratory disturbance; and severe, with respiratory disturbance and renal failure. The major complications affecting the prognosis of NMS are respiratory disturbance and renal failure. Renal failure is also associated with the occurrence of disseminated intravascular coagulation and rhabdomyolysis. The proposed classification system for NMS patients is useful in selecting the appropriate therapeutic strategy for this disorder. The clinical data were analyzed to determine the factors in the process of deterioration in NMS.

Abnormal distribution of neurofilament L in neurons with Alzheimer's disease.

Abnormality of cytoskeletal proteins is closely related to the pathology of Alzheimer's disease. As neurofilament proteins are major cytoskeletal components of neurons, abnormality of neurofilaments is proposed in brain with Alzheimer's disease. Free-floating sections of the hippocampus with Alzheimer's disease were studied immunohistochemically, using a polyclonal antibody specifically bound to the tail region of neurofilament L (NF-L). In brains with early onset type of Alzheimer's disease, many neurons and dystrophic neurites were labeled by the antibody, while these observations were not seen in either brains with late onset type or control brains. Double immunohistochemical staining of NF-L and tau protein demonstrated that abnormal deposition of NF-L was not always accompanied with that of tau protein, indicating that the abnormal deposition of NF-L might not occur in parallel with that of tau protein. These observations suggest the involvement of neurofilament proteins on the pathology of Alzheimer's disease in a different way than tau protein.

The effects of repetitive mild brain injury on cytoskeletal protein and behavior.

The purpose of this study was to examine the hypothesis if repetition of mild mechanical brain injury induces the pathological process related to Alzheimer's disease. After defining the magnitude of the subthreshold brain injury which does not induce brain tissue damage by a single hit, the subthreshold mild impact (1.0 atm) was repeated 7 times every 24 h. One week after the last impact, abnormal accumulation of microtubule-associated protein 2 (MAP2) and phosphorylated neurofilament 200 kD (p-NFH) was observed in neuronal perikarya and dendrites. One month after percussion, the number of MAP2-and p-NFH-positive neuronal perikarya was increased and observed in remote areas including the contralateral cortex and the hippocampus. Tau-1 immunoreactivity was increased in deep cortical neurons of the ipsilateral side after dephosphorylation, indicating the accumulation of phosphorylated tau in neuronal perikarya. The abnormal accumulation of cytoskeletal proteins in neuronal perikarya may be due to impaired axonal transport caused by mechanical brain injury. The behavioral study revealed that after repetitive mild percussion, rats show less efficient habituation to a new environment. It is suggested that the repetition of subthreshold mechanical brain injury may trigger cytoskeletal alteration related to neuronal degeneration.

[Individual differences in the sensitivity to the effect of alcohol upon pain].

In this study, the relationship between pain threshold and blood ethanol level, and also the effect of alcohol sensitivity upon the threshold was investigated. Thirty-one healthy subjects, aged 20 to 62 years, were studied. The genotypes at the aldehyde dehydrogenase 2 (ALDH) locus were determined in each subject to evaluate the alcohol sensitivity. The subjects were divided into three groups based on the ALDH2 genotypes, i.e., the ALDH2*1/*1 (homozygous active ALDH), the ALDH2*1/*2 (heterozygous inactive ALDH) and the ALDH2*2/*2 (homozygous inactive ALDH). The subjects were given orally 0.4 g.kg-1 and/or 0.8 g.kg-1 of ethanol. Venous blood samples were collected at 30, 60, 120, 180, and 240 min after ethanol intake and blood ethanol and acetaldehyde levels were determined. Prior to the ethanol intake and at each sampling of the blood, the pain thresholds were measured by three stimulating methods, i.e., a mechanical method (algometer), a thermal method (Nakahama's pain meter NYT-5) and a chemical method (potassium iontophoretic meter). In the ALDH2*1/*1 and ALDH2*1/*2 groups, the pain threshold measured by the three methods increased with the elevation of blood ethanol levels, especially the threshold by the mechanical method showed clear rise with blood ethanol level. The subjects who belonged to the ALDH2*2/*2 group showed a high blood acetaldehyde level and various psychophysical symptoms. The results of the three tests had no relationships with the variation of blood ethanol levels.

Microbial mutagenicity and in vitro chromosome aberration induction by FK973, a new antitumor agent.

The genotoxic activity of a new antitumor agent, FK973, was compared with that of mitomycin C (MMC) in eukaryotic and prokaryotic cells. In chromosome aberration tests using Chinese hamster fibroblast Don cells, FK973 induced a dose-related increase of aberrant cells after 6 h-pulse treatments, and the minimum effective concentrations with and without S9 were 0.625 and 0.0625 micrograms/ml, respectively. The compound increased revertant colonies in Salmonella typhimurium TA102 at the dose range of 10-5000 micrograms/plate with S9. Without S9, FK973 induced a small increase at the dose range of 500-5000 micrograms/plate in two of three independent experiments, but the number of revertant colonies was less than double that of the vehicle control. The compound did not induce any revertant colonies in colonies in S. typhimurium TA100, TA98, TA1535 or TA1537 with or without S9. MMC was confirmed to increase both chromosome aberrations in Don cells and revertant colonies in TA102. The minimum clastogenic and mutagenic concentrations without S9 were 0.0156 microgram/ml and 0.005 microgram/plate, respectively. The results indicate that FK973 needs metabolic activation to induce reverse mutation in prokaryotic cells, but caused chromosome aberrations in mammalian cells without added S9.

Mutagenicity tests of polyoxyethylene hydrogenated castor oil 60 (HCO-60).

The genotoxicity of polyoxyethylene hydrogenated castor oil 60 (HCO-60) was examined in reverse mutation test in bacteria, chromosome aberration test in vitro and micronucleus test in mice. The in vitro tests were done with and without metabolic activation using rat liver microsomal fraction. HCO-60 did induce neither reverse mutation in Salmonella typhimurium TA100, TA98, TA1535, and TA1537 and in Escherichia coli WP2uvrA, nor chromosome aberrations in Chinese hamster V79 cells. In addition, no increase in micronucleated polychromatic erythrocytes was elicited in the bone marrow of BDF1 male and female mice. It is concluded that HCO-60 was not genotoxic in these in vitro and in vivo assays.