Pentachlorophenol inhibits micronuclei induction by 2-acetylaminofluorene but not by thioacetamide

Environmental Toxicology and Pharmacology 21 (2006) 56–60

Pentachlorophenol inhibits micronuclei induction by 2-acetylaminofluorene but not by thioacetamide E. Zamorano-Ponce a,∗ , J. Fern´andez Romero a , P. Rivera Caama˜no a , C. Barrios Guerra b a b

Laboratorio de Gen´etica Toxicol´ogica (GENETOX), Departamento de Ciencias B´asicas, Facultad de Ciencias, Universidad del B´ıo-B´ıo, Casilla 447, Chill´an, Chile Laboratorio de Toxicolog´ıa, Facultad de Farmacia, Universidad de Concepci´on, Casilla 237, Concepci´on, Chile Received 5 May 2004; received in revised form 28 June 2005; accepted 1 July 2005 Available online 12 September 2005

Abstract Our study examined the capacity of pentachlorophenol (PCP) to inhibit the ability of 2-acetylaminofluorene (2-AAF) and thioacetamide (TAA) to induce micronuclei in mouse bone marrow cells in vivo. 2-AAF (5.6 mg/kg) and TAA (60 mg/kg) were administered intra-peritoneally (i.p.) to Mus musculus males (BALB/c), and the frequencies of polychromatic erythrocytes with micronuclei (PCE-MN) 24 h after injection were analyzed. Treatment with 2-AAF or TAA resulted in high PCE-MN frequencies in comparison with untreated and negative controls (19.9 and 21.6‰, respectively, versus ≈3‰). Pretreatment with a single PCP dose (44 mg/kg) 24 h prior to the 2-AAF administration virtually eliminated micronuclei formation by 2-AAF, although it had no inhibitory effect on TAA-induced micronuclei. Animals receiving cyclophosphamide (CP) served as positive control. Since PCP is known to inhibit arylsulfotransferase (AST) activity, which is involved in 2-AAF activation, this mechanism most likely produced the results with PCP and 2-AAF. Our results also are consistent with a different pathway involved in TAA induction of micronuclei, one that is not inhibited by PCP. © 2005 Elsevier B.V. All rights reserved. Keywords: Pentachlorophenol; Micronucleus; Thioacetamide; 2-Acetylaminofluorene; Arylsulfotransferase; ArylSULT

1. Introduction Chlorinated phenols are chlorine-substituted phenols whose biological activity renders them useful in a range of applications from bactericides to insecticides. PCP has been used as an herbicide, algaecide, defoliant, wood preservative, germicide, fungicide and molluscicide (Chhabra et al., 1999). Due to improper disposal, PCP has become an environmental pollutant and is now considered ubiquitous (Wang et al., 2000). It has been found in soil (Guiraud et al., 2003), water (Hoffmann et al., 2003), air (Wilson et al., 2003), food products (Kieszak et al., 2002) and in humans including: blood (Heudorf et al., 2000), urine (Hill et al., 1989; Becker et al., 2003), milk (Ewers et al., 1999) and umbilical cord plasma (Sandau et al., 2002). ∗

Corresponding author. Tel.: +56 42 203075; fax: +56 42 203046. E-mail address: [email protected] (E. Zamorano-Ponce).

1382-6689/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.etap.2005.07.013

During the past 30 years, different studies have been carried out to measure PCP capacity to induce genetic damage. Although some authors established a weak mutagenic effect (Fahrig, 1974; Fahrig et al., 1978; Bauchinger and Premdas, 1988) and clastogenic activity (Ahmad et al., 2002; Ateeq et al., 2002; Farah et al., 2003; Pavlica et al., 2000), others did not observe any cytogenetic effect in different assay systems (Andersen et al., 1972; Vogel and Chandler, 1974; Buselmaier et al., 1973; Monteith, 1992; Zamorano-Ponce et al., 1994; Ress et al., 2002). It has been well documented that PCP is a specific inhibitor of aryl sulfotransferase (AST) activity (Meerman et al., 1981; Lai et al., 1987; Ringer and Norton, 1987; van de Poll et al., 1989), a primary pathway in the metabolic activation of some hepatocarcinogens. On the basis of this knowledge, the aim of this study is to determine if PCP inhibits the micronuclei induction caused by 2AAF. The study compares 2-AAF with TAA, a hepatocarcinogen that does not utilize AST for its metabolic activation. Specifically, the principal objective

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of this study is to examine PCP capacity to inhibit 2-AAF’s and TAA’s ability to induce micronuclei in mouse bone marrow cells, using the mouse micronucleus test, a sensitive and accepted method for detecting genetic damage in vivo.

2. Material and methods 2.1. Chemicals 2-AAF [CAS 53-96-3], PCP [CAS 87-86-5], TAA [CAS 62-55-5], DMSO [CAS 67-68-5], CP [CAS 50-18-0] and fetal bovine serum (FBS) were purchased from Sigma Chemical Company, St. Louis, MO, USA. 2.2. Animals Males, Mus musculus, BALB/c strain, 6–8 weeks old, weighing approximately 25 g were supplied by the Animal House of the Faculty of Science of the Universidad de B´ıoB´ıo. Animal use and care in all experiments comply with Chilean ethical laws on animal manipulation. Mice were housed in plastic cages at 22 ± 1 ◦ C, 60 ± 10% humidity and 12 h light:12 h dark cycle during the acclimatization period (1 month) to laboratory conditions and throughout the entire experimental period. The mice were fed a balanced diet (Kimber® chow) and tap water ad libitum. 2.3. Treatments 2-AAF, TAA and PCP treatment doses were experimentally determined following the method proposed by Weil (1952). 2-AAF was dissolved in DMSO and used at a concentration of 5.6 mg/kg. TAA was dissolved in FBS and used at a concentration of 60 mg/kg (80% LD50/7 ), and PCP was dissolved in corn oil and used at a concentration of 44 mg/kg mouse body weight (80% LD50/7 ), approximating the maximum tolerated dose according to the criteria proposed by Salamone and Heddle (1983). The positive control group was administered cyclophosphamide (40 mg/kg). All the treatments were intra-peritoneal (i.p.) and the volume of each treatment was maintained uniformly: 0.5 ml/20 g mouse body weight by diluting the chemicals accordingly. Animals in each negative control group were dosed with an equivalent volume of FBS, corn oil, DMSO, corn oil + DMSO or corn oil + FBS.

drop was smeared on a slide and then air-dried. Preparations were stained with Giemsa following the method proposed by Gollapudi and Kamra (1979). Micronuclei were scored at 24 h following the last treatment. At least 2000 tinge-blue colored polychromatic erythrocytes (PCEs) were scanned for eight animals per group. The number of micronucleus (MN) for each one was recorded, and the number of MN per thousand PCE was calculated. Precautions with regard to scoring and artifacts were taken: the slides were scored without prior knowledge of the treatment group (blind) and the micronuclei were scored from PCEs. The results were statistically evaluated using the student’s t-test. To evaluate any perturbation (cytotoxicity) in hematopoiesis as a result of treatment, the number of normochromatic erythrocytes (NCE) in 200 PCE (PCE/NCE ratio) in bone marrow preparations was also analyzed. The sample size of 200 cells is adequate according to the criteria proposed by Gollapudi and McFadden (1995).

3. Results Table 1 shows the PCE-MN frequencies at 24 h in untreated and treated groups. Data (not shown) indicate that the PCE-MN frequencies obtained in the negative controls (i.e., animals dosed with the vehicles: FBS, corn oil, DMSO, corn oil + DMSO or corn oil + FBS) are not statistically different from the “spontaneous frequency” obtained in historical standard untreated laboratory animal stocks (3.2 ± 0.9). The PCP-treated group presented no statistical difference from its respective control group dosed with corn oil at any sample time. A significant increase (p ≤ 0.01) in the PCE-MN frequency over its respective negative control (BFS or DMSO) was observed 24 h after i.p. injection with TAA (60 mg/kg) or 2-AAF (5.6 mg/kg). Pretreatment with PCP 24 h prior to 2-AAF administration almost completely prevented micronuclei formation induced by this arylamine. Micronuclei frequency in this experimental condition is only slightly higher than that of the negative control (oil), but statistically different from the group treated only with the hepatocarcinogen. The values concerning the ratio PCE/NCE indicates that the lower frequency of PCP-MN observed in the group pretreated with Table 1 Frequency of micronucleated polychromatic erythrocytes (PCE-MN) in mice treated with thioacetamide (TAA) or 2-acetylaminofluorene (2-AAF) and the effect of a 24 h PCP pretreatment Pretreatment treatment

No. of total PCE (no. of mice)

No. of PCE-MN*

PCE/NCE*

None PCP None PCP (−24 h) None PCP (−24 h)

16000 (8) 16000 (8) 16000 (8) 16000 (8) 16000 (8) 16000 (8)

3.2 ± 0.9 3.5 ± 1.2 21.6 ± 1.1a 20.9 ± 0.9a 19.9 ± 0.8a 6.4 ± 0.3b

1.7 ± 0.11 1.9 ± 0.19 1.7 ± 0.5 1.9 ± 0.3 2.1 ± 0.2 1.9 ± 0.01

2.4. Micronucleus assay Mice were sacrificed by cervical dislocation and the femur removed by trimming away the skin and muscle and cutting at the pelvic socket and knee joint. The proximal end of the femur was shortened with scissors to obtain a small opening through which bone marrow was flushed into a syringe containing FBS (0.15 ml). The suspension was carefully mixed to obtain a homogeneous, representative sample and a little

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a b *

None None TAA TAA 2-AAF 2-AAF

Significantly different from untreated animals at p ≤ 0.01. Significantly different from animals treated with 2-AAF alone (p ≤ 0.01). Values are mean ± standard error of the mean.

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PCP and treated with 2-AAF was not a result of less proliferate activity in this group when compared to the group treated only with 2-AAF. No statistical differences were found between animal treated with TAA or PCP + TAA. In this respect, the results of other experimental designs were examined, such as coadministration of PCP together with hepatocarcinogens and protocols in which PCP was given at 12 and 48 h before the administration of the hepatocarcinogens. These results indicated a maximum inhibition of micronuclei formation when PCP was given 24 h prior the 2-AAF administration. A positive response in terms of micronucleus formation inhibition in the group pretreated with PCP and treated with TAA was not observed in any of these experimental designs.

4. Discussion It has become well established that both mammalian and plant systems can metabolize foreign chemicals by which several innocuous chemical species (i.e. promutagens) can be enzymatically bio-transformed into chemical forms that can damage DNA. Several studies have demonstrated that cytosolic sulfotransferases enzymes play a fundamental role in the detoxification, metabolism and bioactivation of numerous xenobiotics, many dietary and environmental mutagens, drugs, neurotransmitters and hormones (Peng et al., 2003). These reactions involve the transfer of the sulfuryl group from 3 -phosphoadenosine 5 phosphate (PAPS) to the hydroxyls/amino groups of acceptor molecules, thereby forming sulfuric acid esters and sulfamates (Banoglu, 2000). Concerning the PCP effect on these metabolic activation pathways, it has been shown that the administration of some hepatocarcinogens to rats induces a severe loss of AST activity, which is thought to be a primary pathway in the metabolic activation of various hepatocarcinogens, developing into forms that act as ultimate carcinogens in chemical hepatocarcinogenesis (Ringer and Norton, 1987). In an earlier work, we demonstrated that the pre-treatment with PCP followed by benzidine (BZ) treatment eliminated BZ’s capacity to induce micronuclei in mouse bone marrow erythroblast cells (ZamoranoPonce et al., 1994). As proposed in that paper, this phenomenon could be due to the specific PCP inhibition of the AST activity necessary for the metabolic activation of BZ. That study demonstrated that PCP is unable to induce micronuclei in male mouse bone marrow erythroblast cells. Neither one nor two treatments spaced at 24 h at a concentration of 44 mg/kg (80% LD50/7 ) induced micronuclei. The confirmation of these data in the present study suggests that under our experimental conditions, PCP is unable to induce genetic damage or exclusion of whole chromosomes due to mitotic spindle misfunction. In fact, the values at 30 h for animals dosed with one i.p. PCP injection are not statistically different from those obtained in the negative control (i.e., the group of animals dosed with corn oil and from untreated animals; historical controls, 3.2 ± 0.9).

Other authors working with different experimental systems have demonstrated that PCP was able to induce micronuclei in zebra mussel and great ramshorn snail (Pavlica et al., 2000), Allium cepa root tips (Ateeq et al., 2002) and freshwater fish Channa punctatus (Farah et al., 2003). The MN induction described by these authors could not be confirmed in the present study using the micronucleus test as genetic end-point, as done in a mammalian experimental system. Although we do not have a final explanation, several experimental variables can explain the differences in results obtained with respect to PCP genotoxicity. Confidence in the genetic end point used is an important factor that needs to be considered. The mouse micronucleus test has been used for more than 30 years as an in vivo cytogenetic test to estimate the clastogenic potential of chemicals, becoming a well established, standardized assay. In our opinion, the methodology used, inter-individual variability and the tested compound’s source and purity are also variables that could explain such differences. Methodological aspects mainly include the PCP concentration utilized, and the solubilization form of PCP. Additionally, when animals are used, the number, sex and age of the animals, exposure time, number of − and + controls, data on historical standard untreated laboratory animals stocks, and sample time are also important variables that could explain such differences. The present study reduced the sampling error by analyzing a significant number of animals (eight per group). However, the experimental design also considered other sources of variations that could increase in importance, such us inter-individual differences in metabolic capacity, intoxication and detoxification of the tested compound. Still, if an inbred strain of mouse is used and the methodological variation is minimized (this study), the inter-individual variation should be small. PCP purity could be another parameter that induces a false positive response. Dioxins are produced during PCP chemical synthesis, and consequently when a technical grade PCP or Na-PCP is employed, or when complex mixtures (river water) are tested, cells could also be exposed to these contaminants that effectively induce genetic damage. It has been claimed that most of the toxicity associated with the feeding of technical grade PCP to rats at concentrations of 100 and 20 ppm stems from toxic contaminants rather than from the PCP itself (Kimbrough, 1978). This study used PCP at the highest available purity level, and followed all the recommendations suggested to improve the technique’s accuracy. Given that we have again obtained negative results in terms of PCP micronucleus induction in mouse bone marrow cells, more studies on PCP’s potential to induce genetic damage in eukaryotic cells should be pursued. Data shown in Table 1 also indicate that a single treatment with a low TAA or 2-AAF dose induces a significant increase in PCE-MN frequencies in male mouse bone marrow erythroblast cells and that PCP pretreatment prevents this phenomenon only in the case of 2-AAF. Our data on the PCE/NCE ratio discard the possibility that low micronuclei frequencies found in the groups pretreated with PCP and

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treated with 2-AAF could be due to an impaired cell proliferation. Our findings concerning the two distinct responses to PCP pretreatment for the carcinogens tested confirm that at least two mechanisms are at work in the micronuclei induction in mouse bone marrow cells: (i) a mechanism involving an inhibited AST pathway and (ii) a mechanism independent of AST activity. In the former case, PCP could inhibit the formation of the highly reactive sulfuric acid ester (N-sulfo¨oxy-AF), which is the principal final electrophilic, genotoxic metabolite involved in BZ and 2-AAF metabolic activation (Ringer and Norton, 1987). In the second case, another pathway must be involved in TAA micronuclei induction. It has been shown that thioacetamide requires metabolic activation by one of the P450 enzyme systems (Kim et al., 2000), and probably by both P450 (CYP2E1) and FMO systems (Fort et al., 2003). However, the metabolic pathway to activate thioacetamide into its immunosuppressive form(s) in BALB/c or form(s) that induce teratogenesis in frog embryo may not necessarily be the same one that exerts genetic damage expressed as micronuclei in PCEs. Oxidative stress can be involved as TAA-induced genetic damage. This process could be initiated by thioacetamide-S-oxide, which is derived from TAA biotransformation by the microsomal flavine-adenine dinucleotide (FAD) containing monooxygenase (FMO) and cytochrome P450 systems (Low et al., 2004). ThioacetamideS-oxide is eventually an obligatory intermediate in the metabolic activation of thioacetamide to a reactive metabolite(s), which covalently binds to DNA. This binding could lead to DNA damage in proliferating blast cells, which is expressed as a micronucleus later in the PCE cell population. However, further studies are required to determine the pathway by which TAA induces the apparition of micronuclei in mouse bone marrow cells. Finally, the present study also confirms that the bone marrow micronucleus assay can effectively be used to study the modulation of genetic damage by specifically interfering with a metabolic activation pathway as reported here.

Acknowledgments We gratefully acknowledge Mr. Gerardo F. Quezada Silva for his excellent technical assistance. Financial supports by the Grant No. 012407-2 from the Universidad del B´ıo-B´ıo, Chile is also gratefully acknowledged.

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