Hotaka Saito

Cytotoxicity of 109 chemicals to goldfish GFS cells and relationships with 1-octanol/water partition coefficients

Abstract The midpoint cytotoxicity (NR50 values) of 109 chemicals including alcohols, aromatics, phenols and pesticides to goldfish scale GFS cells were compared with the fish acute toxicities and with the 1 - octanol/water partition coefficient (Pow). The NR50 values were significantly correlated to the available acute toxicity data for fathead minnow (n=31, r=0.958, s=0.480, F=325) and guppy (n=29, r=0.957, s=0.482, F=294). The Pow gave a good correlation in simple linear regression analysis for all compounds excluding 8 compounds that the NR50 values could not be obtained for their low solubility in culture medium. Log 1 NR 50 (mM) = (0.87±0.08) Log Pow − (2.17±0.24) ( n =101, r =0.914, s =0.587, F =505) For 29 narcotic type of compounds, the regression analysis revealed a high correlation between the NR50 values and Pow, and appeared to give a baseline cytotoxicity. Log 1 NR 50 (mM) = (0.77±0.06) Log Pow − (2.48±0.12) ( n =29, r =0.984, s =0.252, F =817) And also, the regression analyses between NR50 and Pow for some types of compounds were discussed. These results suggest that NR assay is useful for prediction of acute toxicity to fish and studies of quantitative structure-activity relationships (QSARs).

Embryonic development assay with Daphnia magna: application to toxicity of aniline derivatives.

An assay system using Daphnia magna embryos was applied to investigate the adverse effects of aniline derivatives. The data were compared with our previous data for chlorophenols. This new assay provides useful information to evaluate the toxicity of chemicals and the differences in sensitivity between the life stages. The effects of 15 aniline derivatives on embryonic development of D. magna embryos were determined. At the start of exposure, 2-6-h old eggs (between stages 1 and 2, round in shape, diameter approx. 400 microm), were used. In control and solvent control groups, embryonic development from an egg to a free-swimming animal proceeded completely within 3 days with more than 90% hatchability. Median effective concentrations (EC50s) to reduce the numbers hatched were determined and gross morphological abnormalities of hatched animals recorded. Anilines induced no obvious morphological abnormalities and no developmental delay although premature deaths occurred. However, they affected the number of embryos hatching in a dose-dependent manner. In addition, this embryo assay was more sensitive to aniline derivatives (except for aniline) than acute juveniles immobilization assay. Ratios of 48-h EC50 (juvenile)/3-day EC50 (embryo) for eight anilines were greater than 5.0. Particularly, the ratios of 4-methyl-, 4-ethyl- and 3-methylaniline were 77, 23 and 11, respectively. EC50s for embryos and juveniles were poorly correlated (r = 0.41). This indicated that the sensitivities of the two life stages were different to the effects of anilines. EC50s were poorly correlated (r = -0.097) with the log Kow (1-octanol/water partition coefficient). These results were compared with previous results for phenols.

In vitro cytotoxicity of 45 pesticides to goldfish GF-scale (GFS) cells

Abstract The cytotoxicities of 45 pesticides including 18 organophosphates and 7 carbamates to GF-Scale (GFS) cells, derived from the scale of goldfish ( Carassius auratus ), were determined with the 24-hr neutral red assay. The assay revealed that 13 pesticides were classified into the highly cytotoxic group (NR50 ≦ 10 mg/L), while 17 pesticides were into the highly cytotoxic group (NR50 ≦ 10 mg/L), while 17 pesticides were moderate (10

Acute toxicity of chlorophenols to earthworms using a simple paper contact method and comparison with toxicities to fresh water organisms.

An acute toxicity test of chlorophenols on earthworms (Eisenia fetida) was performed using a simple paper contact method proposed by OECD testing guideline no. 207, that were applied as an earthworm toxicity test. The median lethal concentration, EC50, had significant correlation with logP(ow) (1-octanol/water partition coefficient) of the chemicals. The toxicity of chlorophenols on E. fetida was compared with toxicities for other species: an algae (Selenostrum capricornutum), a crustacean (Daphnia magna), and a fish (Oryzias latipes). It was found that the toxicity of chlorophenols was almost same for E. fetida and for fresh water organisms. These results suggest the possibility of drawing correlations between the effects of pollutants on living things in different environments, fresh water and soil.

Cytotoxicity of chlorophenols to goldfish GFS cells with the MTT and LDH assays

The cytotoxicity of phenol and 14 chlorophenols (CPs) were examined in goldfish scale (GFS) cells, a fibroblastic cell line derived from goldfish scales. MTT reduction and intracellular LDH activities were examined as cytotoxicity indicators. Cell densities at inoculation were 1.0 x 10(5) cells/ml in the MTT assay and 0.5 x 10(5) or 0.1 x 10(5) cells/ml in the LDH assay. No change in the midpoint cytotoxicity concentration (IC(50)) was observed in either assay at the same order of cell density (0.5-1.0 x 10(5) cells/ml). However, at low chemical concentrations, the LDH assay was more sensitive than the MTT assay. In addition, for many CPs, the LDH assay at 0.1 x 10(5) cells/ml was more sensitive than the MTT and LDH assays at the higher cell density levels. The correlation coefficients (r) for all IC(50) determinations were greater than 0.97. Good correlations between these assays and the neutral red (NR) assay were also observed (r > 0.97). The 1-octanol/water partition coefficient (log P) gave good correlation coefficients (r > 0.90) with IC(50) values of both the MTT and LDH assays in simple linear regression analyses. The study demonstrates that both the MTT and LDH cytotoxicity assays with GFS cells, together with the NR assay, can be applied to screen the acute toxicities of chemicals.

Mutational spectra of benzo[a]pyrene and MeIQx in rpsL transgenic zebrafish embryos

To evaluate the rpsL transgenic zebrafish (Brachydanio rerio) mutation assay, we treated the embryos with benzo[a]pyrene (B[a]P) (10 microg/ml) or 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) (300 microg/ml) for 16h and determined the mutation spectra. These treatments were previously reported to induce mutant frequencies that were 4.3 and 2.4 times the control value, respectively. In the B[a]P-treated group, half of the mutations were single base substitutions, 74% of which occurred at G:C base pairs. Among G:C base pair substitutions, G:C to T:A and G: C to C:G transversions were predominant, suggesting that B[a]P induced mutations in zebrafish embryos by mechanisms previously described in mammalian tissues. In the MeIQx-treated group, about 60% of the mutations were deletions. Some specific mutations were found, but the compound primarily amplified the background mutation level; improvement in the conditions of treatment may be required for elucidating MeIQx-mutagenesis in this system. This study showed that transgenic zebrafish may be a useful tool for detecting mutagens in aquatic environments and for elucidating mutagenic mechanisms.

Cytotoxicity of anilines and aldehydes to goldfish GFS cells and relationships with 1-octanol/water partition coefficients

Abstract The cytotoxicites of 13 anilines and 10 aldehydes to goldfish scale GFS cells were determined with the neutral red assay. The sequence of cytotoxicity was based on the concentration of chemicals that reduced uptake of neutral red by 50 % (NR50 values). Among a series of aniline derivatives, the parent aniline molecule was the least cytotoxic, with potency increasing with the progressive incorporation of chlorine atoms or with the alkyl chain length into the aromatic ring structure. Among a series of aldehyde derivatives, methanal (formaldehyde) and propanal, which have less than two carbon atoms of the alkyl chains, were more cytotoxic than predicted from the alkyl chain length. The in vitro cytotoxicity of these chemicals except for methanal was found to be significantly correlated to their in vivo acute toxicity to guppies. Moreover, NR50 values were significantly correlated with 1-octanol/water partition coefficients (Pow) excluding the lower aliphatic aldehydes.