Frameshift mutations induced by the acridine mustard ICR-191 in embryos and in the adult gill and hepatopancreas of rpsL transgenic zebrafish

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Abstract

To determine whether frameshift mutations can be detected in rpsL transgenic zebrafish (Brachydanio rerio), embryos, and adult fish were treated with 6-chloro-9-[3-(2-chloroethylamino)-propylamino]-2-methoxyacridine (ICR-191). Embryos exposed to 0, 10, or 20 μM ICR-191 in a water bath for 18 h exhibited induced mutant frequencies (MFs) of 14 × 10−5, 16 × 10−5, and 25 × 10−5, respectively. Only embryos exposed to 20 μM ICR-191 showed a significant increase in MF. The mutational spectra differed between the control and ICR-191-treated groups and single G:C pair insertions, which are a marked characteristic of ICR-191 mutagenesis, were observed in both 10 and 20 μM-treated embryos. In adult fish treated with 1 μM ICR-191 in a water bath for 18 h, a significant increase in MFs was observed in both gill (12 × 10−5 and 44 × 10−5 in control and treated fish, respectively), and hepatopancreas (5 × 10−5 and 29 × 10−5, respectively) 2 weeks after exposure. Sequence analysis showed that 58% of mutations in gill and 94% of mutations in hepatopancreas were single G:C pair insertions, which is typical of mutations induced by ICR-191. Additionally, these mutations occurred predominantly at a single site (CC sequence at bps 140–141) in the rpsL gene. Three weeks after exposure, however, the increased MFs and prominent mutational spectra of ICR-treated fish were undetectable. These findings suggest that using our protocols the rpsL transgenic zebrafish mutation assay is more effective for adult fish than for embryos, but that frameshift mutations can be detected in both embryos and adults at appropriate sampling times after treatment with ICR-191.

Introduction

Water pollution is a serious problem for aquatic species and, indeed, for all animals including humans, via exposure through drinking water. Among the pollutants, genotoxic substances are extremely deleterious, because DNA lesions induced by such chemicals can cause tumors and/or be inherited across generations if the lesions occur in germ-line cells. Water contaminants often contain many unknown compounds, and therefore several bioassay methods [1], such as the Ames test [1], the micronucleus test [2], [3], and the comet assay [4], [5], have been used to detect water-borne genotoxicity.

The Ames test, which has been the most widely used, measures induced mutagenicity in Salmonella. In order to assess environmental risk, bioassay systems for detecting in vivo mutagenicity in higher organisms is required. Development of transgenic rodent mutation assay systems with recoverable target genes have made it possible to determine in vivo mutagenicity. The transgenic rodent system has been extensively used and its usefulness in predicting carcinogenicity has been demonstrated [6], [7].

Similarly, fish could provide an in vivo system advantageous for detecting mutagenicity, since fish have proven to be a useful model of chemical carcinogenesis. A fish mutagenicity system has other advantages compared to the rodent system: fish are easier to expose to complex mixtures of environmental samples for long periods, they are easier to maintain at lower costs, and require less space. Additionally, fish embryos can be exposed to compounds much more easily than rodent embryos because fish eggs are externally fertilized and undergo development in the aquatic environment. Moreover, it is important to assay mutagenicity in aquatic species to predict ecological hazard in aquatic environments. Accordingly, we have developed a transgenic zebrafish (Brachydanio rerio) line carrying the rpsL gene as a recoverable mutational target [8]. A transgenic Medaka (Oryzias latipes) carrying the λcII target gene was recently developed for similar assays [9].

We first used embryos of the rpsL transgenic zebrafish and showed that N-ethyl-N-nitrosourea (ENU) [8] and benzo[a]pyrene [10] induce typical base substitution mutations. Recently, we successfully developed an assay using adult rpsL fish and showed that N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) induces typical base substitution mutations in the gill and hepatopancreas of both adults and embryos [11]. To use the rpsL transgenic zebrafish in environmental risk assessment, further characterization, such as detection of mutagenicity of various well-known mutagens is required. Recently, Ohe et al. have reviewed mutagenic/genotoxic bioassay data published since 1990 and reported that the percentage classified as highly/extremely mutagenic was 3–5% both towards frameshift and base substitute type Salmonella by the Ames test [1]. We have already shown that the rpsL zebrafish can quantitatively detect base substitution mutations [11], and here we intended to determine whether rpsL transgenic zebrafish are suitable for the detection of frameshift mutations. For this purpose, the acridine mustard 6-chloro-9-[3-(2-chloroethylamino)-propylamino]-2-methoxyacridine (ICR-191) (Fig. 1) was chosen, since this compound induces predominantly single base insertions and/or deletions in various systems [12], [13], [14], [15], [16], [17], [18].

Acridine compounds are planar polycyclic aromatic molecules that bind DNA tightly but reversibly by intercalation [19]. Among various acridine compounds, ICR-191 is considered to be more biologically active than the acridine moiety alone, because it contains an alkylating nitrogen mustard side chain [19]. Acridine compounds are well known as dyes and nucleic acid stains, and they are also medically important as antibacterial and anticancer agents. The acridines are azaarene compounds, nitrogen in-ring substituted polycyclic aromatic hydrocarbons, and have been found in a range of environmental contaminants derived from similar sources to those containing polyaromatic hydrocarbons. Because of its reactive mustard chain, ICR-191 may not exist in the environment for long periods of time; however, acridine itself and other acridine derivatives have been detected in aquatic environments [20], [21], [22], [23], [24]. As a model acridine derivative, ICR-191 may be important for characterizing the usefulness of our fish mutagenicity system in environmental risk assessment.

In this study, characteristic mutations of ICR-191, single G:C base pair insertions, were detected in both embryos and adult tissues. The increase in MFs was marked in adult fish at 2 weeks after the treatments, but disappeared by 3 weeks. The reason for this disappearance and the importance of determining an optimal post-exposure sampling time will be discussed.

Section snippets

Transgenic zebrafish, competent cells, and chemicals

A transgenic zebrafish line carrying approximately 350 copies of the shuttle vector pML4 per haploid genome [8] has been maintained in rearing water (0.1% Instant Ocean salts in reverse osmotic and deinonized water) at 26 °C over several generations. Escherichia coli (E. coli) RR1 was obtained through the Japanese Cancer Research Resources Bank. ICR-191 (dihydrochloride salt, CAS Registry No. 17070-45-0), dimethyl sulfoxide (DMSO, CAS No. 67-68-5, >99.5%), and N-2-hydroxyethylpiperazine-N

MF and mutational spectra induced by ICR-191 in embryos

To examine whether ICR-191 mutagenicity can be detected by our protocol using embryos of rpsL transgenic zebrafish, pfd 1 embryos (about 29 pfh) were exposed to 0, 10, or 20 μM ICR-191 in a water bath for 18 h, and a mutation assay was performed at pfd 4 (2 days after the end of the treatments). One hundred and fifty embryos per group were used to obtain one MF and three replicate groups were exposed at each dose, as described in Section 2. The survival and the developmental status of

Mutagenicity assay using rpsL transgenic zebrafish embryos

The marked advantage of a fish embryo assay system compared to the rodent system is that quantitative exposures are more readily achieved since fertilization and development take place externally, and, therefore, water-borne exposure is possible. Further, only small volumes (i.e.10 mL) of solution are sufficient for exposure of each group, and, therefore, only small amounts of chemicals are required. Additionally, repeated breeding of the same founder fish is possible.

A disadvantage of our

Acknowledgements

We thank Keiko Miki for her excellent technical contribution. We also thank Tamaki Takahashi for maintaining fish, Dr. Haruki Tatsuta for his help in statistical analysis and Akiko Hashimoto for helpful discussion.

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