Shuji Tsuda

The comet assay with 8 mouse organs: results with 39 currently used food additives

We determined the genotoxicity of 39 chemicals currently in use as food additives. They fell into six categories-dyes, color fixatives and preservatives, preservatives, antioxidants, fungicides, and sweeteners. We tested groups of four male ddY mice once orally with each additive at up to 0.5xLD(50) or the limit dose (2000mg/kg) and performed the comet assay on the glandular stomach, colon, liver, kidney, urinary bladder, lung, brain, and bone marrow 3 and 24h after treatment. Of all the additives, dyes were the most genotoxic. Amaranth, Allura Red, New Coccine, Tartrazine, Erythrosine, Phloxine, and Rose Bengal induced dose-related DNA damage in the glandular stomach, colon, and/or urinary bladder. All seven dyes induced DNA damage in the gastrointestinal organs at a low dose (10 or 100mg/kg). Among them, Amaranth, Allura Red, New Coccine, and Tartrazine induced DNA damage in the colon at close to the acceptable daily intakes (ADIs). Two antioxidants (butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT)), three fungicides (biphenyl, sodium o-phenylphenol, and thiabendazole), and four sweeteners (sodium cyclamate, saccharin, sodium saccharin, and sucralose) also induced DNA damage in gastrointestinal organs. Based on these results, we believe that more extensive assessment of food additives in current use is warranted.

The Comet Assay with Multiple Mouse Organs: Comparison of Comet Assay Results and Carcinogenicity with 208 Chemicals Selected from the IARC Monographs and U.S. NTP Carcinogenicity Database

ABSTRACT The comet assay is a microgel electrophoresis technique for detecting DNA damage at the level of the single cell. When this technique is applied to detect genotoxicity in experimental animals, the most important advantage is that DNA lesions can be measured in any organ, regardless of the extent of mitotic activity. The purpose of this article is to summarize the in vivo genotoxicity in eight organs of the mouse of 208 chemicals selected from International Agency for Research on Cancer (IARC) Groups 1, 2A, 2B, 3, and 4, and from the U.S. National Toxicology Program (NTP) Carcinogenicity Database, and to discuss the utility of the comet assay in genetic toxicology. Alkylating agents, amides, aromatic amines, azo compounds, cyclic nitro compounds, hydrazines, halides having reactive halogens, and polycyclic aromatic hydrocarbons were chemicals showing high positive effects in this assay. The responses detected reflected the ability of this assay to detect the fragmentation of DNA molecules produced by DNA single strand breaks induced chemically and those derived from alkali-labile sites developed from alkylated bases and bulky base adducts. The mouse or rat organs exhibiting increased levels of DNA damage were not necessarily the target organs for carcinogenicity. It was rare, in contrast, for the target organs not to show DNA damage. Therefore, organspecific genotoxicity was necessary but not sufficient for the prediction of organ-specific carcinogenicity. It would be expected that DNA crosslinkers would be difficult to detect by this assay, because of the resulting inhibition of DNA unwinding. The proportion of 10 DNA crosslinkers that was positive, however, was high in the gastrointestinal mucosa, stomach, and colon, but less than 50% in the liver and lung. It was interesting that the genotoxicity of DNA crosslinkers could be detected in the gastrointestinal organs even though the agents were administered intraperitoneally. Chemical carcinogens can be classified as genotoxic (Ames test-positive) and putative nongenotoxic (Ames test-negative) carcinogens. The Ames test is generally used as a first screening method to assess chemical genotoxicity and has provided extensive information on DNA reactivity. Out of 208 chemicals studied, 117 are Ames test-positive rodent carcinogens, 43 are Ames test-negative rodent carcinogens, and 30 are rodent noncarcinogens (which include both Ames test-positive and negative noncarcinogens). High positive response ratio (110/117) for rodent genotoxic carcinogens and a high negative response ratio (6/30) for rodent noncarcinogens were shown in the comet assay. For Ames test-negative rodent carcinogens, less than 50% were positive in the comet assay, suggesting that the assay, which detects DNA lesions, is not suitable for identifying nongenotoxic carcinogens. In the safety evaluation of chemicals, it is important to demonstrate that Ames test-positive agents are not genotoxic in vivo. This assay had a high positive response ratio for rodent genotoxic carcinogens and a high negative response ratio for rodent genotoxic noncarcinogens, suggesting that the comet assay can be used to evaluate the in vivo genotoxicity of in vitro genotoxic chemicals. For chemicals whose in vivo genotoxicity has been tested in multiple organs by the comet assay, published data are summarized with unpublished data and compared with relevant genotoxicity and carcinogenicity data. Because it is clear that no single test is capable of detecting all relevant genotoxic agents, the usual approach should be to carry out a battery of in vitro and in vivo tests for genotoxicity. The conventional micronucleus test in the hematopoietic system is a simple method to assess in vivo clastogenicity of chemicals. Its performance is related to whether a chemical reaches the hematopoietic system. Among 208 chemicals studied (including 165 rodent carcinogens), 54 rodents carcinogens do not induce micronuclei in mouse hematopoietic system despite the positive finding with one or two in vitro tests. Forty-nine of 54 rodent carcinogens that do not induce micronuclei were positive in the comet assay, suggesting that the comet assay can be used as a further in vivo test apart from the cytogenetic assays in hematopoietic cells. In this review, we provide one recommendation for the in vivo comet assay protocol based on our own data.

Detection of chemically induced DNA lesions in multiple mouse organs (liver, lung, spleen, kidney, and bone marrow) using the alkaline single cell gel electrophoresis (Comet) assay

The effect of 2 model chemical mutagens on DNA was evaluated with the alkaline single cell gel electrophoresis (SCG) (Comet) assay in 5 mouse organs--liver, lung, kidney, spleen and bone marrow. Mice were sacrificed 3 and 24 h after the administration of the direct mutagen ethyl nitrosourea (ENU) or the liver-targeting promutagen p-dimethylaminoazobenzene (DAB). Each organ was minced, suspended at a concentration of 1 g/ml in chilled homogenizing buffer (pH 7.5) containing 0.075 M NaCl and 0.024 M Na2EDTA, homogenized gently using a Potter-type homogenizer at 500-800 rpm set in ice, and then centrifuged nuclei were used for the alkaline SCG assay. ENU induced DNA damage in cells all of the organs studied DAB, on the other hand, produced a positive response in the liver only. We suggest that it may be possible to use the alkaline SCG assay using a homogenization technique to detect the genotoxicity of chemicals in vivo in their target organs.

PFOS and PFOA in environmental and tap water in China

There is a great concern about global contamination with persistent fluoroorganic compounds including perfluorooctane sulfonate (PFOS) and perfluorooctanoate (PFOA), however, few data are available on the environmental levels of these chemicals in China. In the present study, therefore, environmental or tap water samples collected from various regions of China were assayed for PFOS and PFOA by solid phase extraction and liquid chromatography-mass spectrometry technique. Median concentrations (maximum concentration) of PFOS and PFOA in environmental water were 0.4 (2.4) and 0.1 (1.3) ngL(-1) for the remote area (n=13), 4.0 (14.1) and 3.9 (30.8) ngL(-1) for the urban area (n=22), respectively. Systematic survey was also conducted in the Hun River (n=11) and the Yangtze River (n=34). In the Hun River, the median of PFOS concentration was 4.9ngL(-1), while PFOA was below the limit of quantitation (0.1ngL(-1)) at many of the sampling sites. The Yangtze River was moderately contaminated with both chemicals: median concentration was 4.2ngL(-1) for PFOS and 5.4ngL(-1) for PFOA. Remarkably high concentration of PFOA was found at 2 sampling sites of the Yangtze River (110.6 and 297.5ngL(-1)), but the concentration had declined to the average level at the next sampling site in both cases. Many cities provided tap water with low levels of PFOS and PFOA, however, tap water in Guangzhou and Shenzhen exceeded 10ngL(-1) for both chemicals. This study revealed obvious presence of perfluorinated compounds spread out the entire territory of China, and the levels in urban area of China were almost comparable to those in the US, Europe and Japan.

Simple detection of chemical mutagens by the alkaline single-cell gel electrophoresis (Comet) assay in multiple mouse organs (liver, lung, spleen, kidney, and bone marrow)

Recently, we designed a fast and simple method to obtain nuclei for the alkaline SCG assay and we tested it with mouse liver, lung, kidney, spleen, and bone marrow. Instead of isolating organ cells by trypsinization, we homogenized tissue and isolated the nuclei. Each organ was minced, and the mince was suspended in chilled homogenizing buffer containing NaCl and Na2EDTA, homogenized gently using a Potter-type homogenizer set in ice, and then centrifuged. The nuclei from the precipitate were used for the assay. To evaluate the validity of this method, we tested the genotoxicity in mouse organs of 11 chemical mutagens with different modes of action. Mice were sacrificed 3 and 24 h after administration of each mutagen. Treatment with three alkylating agents (MMS, EMS, and MNNG), a DNA crosslinking agent (MMC), two aromatic amines (2-AAF and phenacetin), a polycyclic aromatic hydrocarbon (B[a]P), and two inorganic chemicals (KBrO3 and K2CrO4) increased migration of the DNA from mouse organs. 5-FU (a base analog) and colchicine (a spindle poison) treatment produced negative results in all organ studied. Considering that the alkaline SCG assay detects genotoxicity as DNA fragments derived from DNA single-strand breaks and alkali-labile damage, our results showed that the SCG assay using our homogenization technique detected chemical mutagens as a function of their modes of action.

Perfluorinated Compounds in the Environment and the Blood of Residents Living near Fluorochemical Plants in Fuxin, China

A fluorochemical industrial park was built in 2004 in Fuxin, China, for the production of polytetrafluoroethylene (PTFE) and perfluorobutane sulfonate (PFBS). Yet little is known about the distribution of fluorochemicals in the environment and in people living in and around the park. In this study, environmental samples were collected from 22 sites in Fuxin to investigate the extent of perfluorinated compound (PFC) contamination in the environment around the park, and in drinking water from the public water supply system and groundwater in shallow aquifers from private wells near the park. Serum samples were also collected from nonoccupationally exposed residents living in Fuxin to determine the PFC load of local residents. As the dominant contaminant of eight target PFCs, the maximum concentrations of perfluorooctanoic acid (PFOA) in sediment and river water of the River Xi along the industrial park were 48 ng/g dry weight and 668 ng/L, respectively; the highest PFOA concentration in groundwater beneath the park was 524 ng/L; and the PFOA levels in drinking water from the public water supply system ranged between 1.3 and 2.7 ng/L. In human serum, PFOA had the geometric mean at 4.3 ng/mL, ranging from 0.02 to 93 ng/mL. This study serves to document what should be the beginning of a long-term surveillance effort to minimize potential exposure of residents living in Fuxin.

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.

Detection of rodent liver carcinogen genotoxicity by the alkaline single-cell gel electrophoresis (Comet) assay in multiple mouse organs (liver, lung, spleen, kidney, and bone marrow)

We have recently designed a simple method for applying the alkaline single-cell gel electrophoresis (SCG) assay to mouse organs. With this method, each organ is minced, suspended in chilled homogenizing buffer containing NaCl and Na2EDTA, gently homogenized using a Potter-type homogenizer set in ice, and then centrifuged nuclei are used for the alkaline SCG assay. In the present study, we used the method to assess the genotoxicity of 8 rodent hepatic carcinogens in 5 mouse organs (liver, lung, kidney, spleen, and bone marrow). The carcinogens we studied were p-aminoazobenzene, auramine, 2,4-diaminotoluene, p-dichlorobenzene, ethylene thiourea (ETU), styrene-7,8-oxide, phenobarbital sodium, and benzene-1,2,3,4,5,6-hexachloride (BHC); except for p-aminoazobenzene, they do not induce micronuclei in mouse bone marrow cells. Mice were sacrificed 3 and 24 h after the administration of each carcinogen. p-Aminoazobenzene, ETU, and styrene-7,8-oxide induced alkaline labile DNA lesions in all of the organs studied. Auramine, 2,4-diaminotoluene, p-dichlorobenzene, and phenobarbital sodium also produced lesions, but their effect was greatest in the liver. BHC, which is not genotoxic in in vitro tests, did not show any effects. We suggest that it may be possible to use the alkaline SCG assay to detect in vivo activity of chemicals whose genotoxicity is not expressed in bone marrow cells.

Detection of in vivo genotoxicity of 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX) by the alkaline single cell gel electrophoresis (Comet) assay in multiple mouse organs

We tested the genotoxicity of 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX) in the mouse in 6 organs (liver, lung, kidney, brain, spleen, and bone marrow) and in the mucosa of stomach, jejunum, ileum, colon, and bladder using the alkaline single-cell gel electrophoresis (SCG) (Comet) assay modified by us. Mice were sacrificed 1, 3, 6, and 24 h after oral administration of the mutagen at 100 mg/kg. MX yielded statistically significant DNA damage in the liver, kidney, lung, and brain and in all the mucosa samples. While DNA damage persisted in the gastrointestinal and urinary tract for 6-24 h after a single oral dosing, it peaked in the liver at 1 h and returned to almost the control level at 3 h. Our present results suggest that MX is genotoxic for various mouse organs, but not for the hematopoietic system, and that the alkaline SCG assay with a homogenization technique can be used to predict genotoxicity in the gastrointestinal and urinary tracts.

Detection of in vivo genotoxicity of endogenously formed N-nitroso compounds and suppression by ascorbic acid, teas and fruit juices

The genotoxicity of endogenously formed N-nitrosamines from secondary amines and sodium nitrite (NaNO(2)) was evaluated in multiple organs of mice, using comet assay. Groups of four male mice were orally given dimethylamine, proline, and morpholine simultaneously with NaNO(2). The stomach, colon, liver, kidney, urinary bladder, lung, brain, and bone marrow were sampled 3 and 24 h after these compounds had been ingested. Although secondary amines and the NaNO(2) tested did not yield DNA damage in any of the organs tested, DNA damage was observed mainly in the liver following simultaneous oral ingestion of these compounds. The administration within a 60 min interval also yielded hepatic DNA damage. It is considered that DNA damage induced in mouse organs with the coexistence of amines and nitrite in the acidic stomach is due to endogenously formed nitrosamines. Ascorbic acid reduced the liver DNA damage induced by morpholine and NaNO(2). Reductions in hepatic genotoxicity of endogenously formed N-nitrosomorpholine by tea polyphenols, such as catechins and theaflavins, and fresh apple, grape, and orange juices were more effective than was by ascorbic acid. In contrast with the antimutagenicity of ascorbic acid in the liver, ascorbic acid yielded stomach DNA damage in the presence of NaNO(2) (in the presence and absence of morpholine). Even if ascorbic acid acts as an antimutagen in the liver, nitric oxide (NO) formed from the reduction of NaNO(2) by ascorbic acid damaged stomach DNA.