DNA Damage and Glutathione Peroxidase Activity in Liver and Kidney Cells in Wistar Rats Exposed to Terbuthylazine (TERB) for 28 Consecutive Days

*Vilena Kašuba, Vedran Micek, Alica Pizent, Blanka Tariba Lovaković, Davor Želježić, Nevenka Kopjar and Mirta Milić*

## **Abstract**

The potential of low doses of the chloro-triazine herbicide terbuthylazine to induce DNA damage and impair activity of glutathione peroxidase (GPx) was evaluated in kidney and parenchymal and non-parenchymal liver cells of adult male rats. In a 28-day study, terbuthylazine was applied daily by oral gavage at doses: 0.004, 0.4 and 2.29 mg/kg bw/day. Tail Intensity (T Int) and Tail Length (TL) were used as descriptors of DNA damage. In the kidney, Tail Int was significantly different in all treated groups, while TL was different in 0.4 and 2.29 mg/kg bw/day groups, compared to controls. Significant differences in TL were recorded in parenchymal and non-parenchymal liver cells of all treated groups. Tail Int was significantly different from controls in non-parenchymal liver cells at all applied doses and in parenchymal cells at terbuthylazine doses of 0.004 and 2.29 mg/kg bw/day. A significant increase in GPx activity was observed only in the kidney at doses 0.4 and 2.29 mg/kg bw/day compared to the controls indicating its possible role in the protection of kidney from free radicals. It appears that repeated exposure to low doses of terbuthylazine could cause DNA instability in kidney cells and in parenchymal and non-parenchymal liver cells in rats.

### **1. Introduction**

Terbuthylazine (TERB) is a chloro-s-triazine herbicide, mostly used for the removal of weeds to protect crops [1]. It is also used as an aquatic herbicide to control submerged and free-floating weeds and algae in fish ponds, swimming pools and reservoirs [2, 3]. It is the most frequently used triazine in Europe in the last two decades [4, 5]. The EFSA (2011) pointed out that TERB poses a high risk to non-target plants in the off-field areas, while the risk for soil micro- and macroorganisms and bees is low. Mammals could be exposed to TERB through oral,

dermal or inhalation routes [6]. The acute toxicity of TERB can be low to moderate, causing slight eye and skin irritation and sensitisation. TERB shows adverse effects on the cellular activity of enzymes such as aromatase, an enzyme which converts androgen to oestrogen [6], and leads to cytotoxicity, as well as affects the functions of the kidney and liver [7, 8].

People are exposed to TERB in several ways, occupationally through inhalation and dermal contact at workplaces and at places where TERB is produced or used. The general population is mostly exposed through ingestion of contaminated drinking water or by dermal contacts [8].

This herbicide persists in the environment and easily moves from treated soil to water [9, 10]. The current cancer classification states TERB belongs to Group D "Not classifiable as a human carcinogen" [7].

Literature data shows that herbicides have the potential to induce reactive oxygen species (ROS), leading to oxidative stress on non-target organisms [11]. The first line of defence against the oxidative stress consists of the antioxidative enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), which convert superoxide anions (O2 − ) into H2O2 and then into H2O and O2 [12–14]. Studies performed in experimental animals showed that atrazine and TERB mostly affected antioxidant defence of exposed animals, depending on species, its sex and age, herbicide concentration, and duration of exposure [15].

An US EPA study [8] indicated that TERB is highly toxic to aquatic organisms. It causes dose-dependent morphological changes and damage in gills, intestine, and kidney of fish [16], and disturbances in biochemical and oxidative stress parameters in carp [17]. Recent studies in aquatic organisms indicated that TERB can induce oxidative stress [18–20] and accumulation of ROS in cells [17, 21–23].

As for TERB genotoxicity, data are limited, i.e. no genotoxic effect was found using the bone-marrow micronucleus test both in female and male mice [24]. In an *in vitro* study, Mladinić et al. [25] showed that TERB could produce primary DNA damage in a 14-day extended human lymphocyte culture. Tariba Lovaković et al. [26] in *in vivo* study on rats exposed orally to TERB points to the disturbance of oxidant/antioxidant balance in erythrocytes and plasma. Plasma SOD activity decreased at two of the smallest doses (0.004 and 0.4 mg/kg bw/day), as well as plasma CAT activity at the highest applied dose (2.29 mg/kg bw/day).

The liver is the primary site of the metabolism, detoxification and excretion of potentially toxic substances [27]. The xenobiotic metabolism starts in the liver immediately after absorption from the gastrointestinal tract. One of the reasons for this is the fact that the liver has the highest supply of biotransformation enzymes of all organs in the body. Literature data [28] point to the fact that the liver is a complex organ with multiple cell types. Hepatocytes represent 60–65% of total rat liver cells [29]. They are responsible for the drug metabolism [30]. Their function is dependent on their micro environment, i.e., of direct cell-cell and cell-matrix interactions, and a lot of diffusible factors secreted by nearby non–parenchymal cells [31]. Non-parenchymal cells represent 40% of the total number of liver cells, but only 6.5% of its volume. These cells contribute to inflammatory responses. Several studies highlighted the importance of non-parenchymal liver cells and their responses to drug toxicity [32–36]. The main process of TERB metabolism in plants and animals is side-chain de-alkylation and oxidation to 2-hydroxy derivatives [37].

The aim of the present study was to investigate the impact of TERB on liver and kidney of adult male Wistar rats treated by oral gavage for 28 consecutive days. We determined levels of primary DNA damage by comet assay measuring Tail Length (TL) and Tail Intensity (T Int) in kidney and two types of liver cells: small or non-parenchymal cells (sized <30 μm head length) and medium sized cells or parenchymal cells or hepatocytes (sized between 30 and 40 μm head length).

**53**

*DNA Damage and Glutathione Peroxidase Activity in Liver and Kidney Cells in Wistar Rats…*

We also measured the GPx activities in liver and kidney tissue to determine the extent of oxidative stress caused by the treatment. The results of the present study could contribute to understanding TERB's potential toxicity related to its low dose

This experiment was conducted at the Institute for Medical Research and

Unit and Analytical Toxicology and Mineral Metabolism Unit, in spring 2016.

anesulfonate (EMS) was purchased from Sigma Aldrich, Germany.

**2.1 Test substance and positive control substance**

Occupational Health (IMROH), Zagreb, Croatia, in Animal Breeding Unit, Mutagenesis

Terbuthylazine (CAS number 5915-41-3), purity grade 99.0%, was purchased as analytical standard from Pestanal® quality (Fluka, Sigma Aldrich, Germany) and dissolved in ethanol (EtOH) to prepare a stock solution. To prepare treatment solutions, the stock solution was diluted with sterile redistilled water. Ethyl meth-

Negative controls received water, whereas positive controls received ethyl methanesulfonate (EMS) at 300 mg/kg bw/day the last three days of the experiment. EMS is a monofunctional alkylating agent recommended for *in vivo* comet assay in rodents [38].

The study was approved by the Ethics Committee of the Institute for Medical Research and Occupational Health (IMROH), Zagreb, Croatia and the Croatian Ministry of Agriculture (Reg.no. 100-21/14-5, Class 01-18/14-02-2/6 of 11 June 2014). Animal treatments were carried out according to internationally accepted animal welfare guidelines [39]. The study was performed using 25 healthy male adult Wistar rats with an initial body weight from 231 g to 271 g. Free access to standard food (Mucedola, 4RF21, Italy) and tap water was ensured. Animals were kept in clear polycarbonate cages with 40–60% humidity at 22°C and normal 12-hr light/dark cycle. At the start of the study, the animals were weighted and inspected

Rats were randomly assigned to the five groups composed of five animals as recom-

mended by JaCVAM (Japanese Center for the Validation of Alternative Methods) [40]. Three groups received TERB at doses of 0.004, 0.4 and 2.29 mg/kg bw/day, respectively, for 28 consecutive days by an oral gavage. These doses were selected based on the reference values set by the EFSA [6]. Negative controls received water, whereas positive controls received ethyl methanesulfonate (EMS) at 300 mg/kg bw/day over the last three days of the experiment. All animals were handled in the same manner. Body weights were regularly monitored (once a week) during the experiment and the doses of TERB were adjusted accordingly. Survival and clinical signs of intoxication were also inspected daily by a licenced veterinarian at IMROH.

The experiment was terminated 24 h after the final gavage. All animals were humanely euthanized by exsanguination under Xylapan/Narketan anaesthesia (Xylapan, Vetoquinol UK Ltd., 12 mg/kg bw *i. p.*/Narketan, Vetoquinol UK Ltd., 80 mg/kg bw) and dissected. Animals were examined for gross pathological

changes of the internal organs by a licenced veterinarian at IMROH.

*DOI: http://dx.doi.org/10.5772/intechopen.94178*

**2. Materials and methods**

exposure.

**2.2 Animals**

by a licenced veterinarian at IMROH.

**2.3 Experimental schedule**

*DNA Damage and Glutathione Peroxidase Activity in Liver and Kidney Cells in Wistar Rats… DOI: http://dx.doi.org/10.5772/intechopen.94178*

We also measured the GPx activities in liver and kidney tissue to determine the extent of oxidative stress caused by the treatment. The results of the present study could contribute to understanding TERB's potential toxicity related to its low dose exposure.
