Effect of arsenic exposure on MDA, SOD, H₂O₂ and TNF-α levels of uterus homogenate of female Sparague-Dawleys rats

Animal model and experimental design

Female Sprague-Dawley rats (Rattus norvegicus L.) aged 6–24 months and weighing 200–300 g were used in this study. The model animals were purchased from the Abadi Jaya Farm in Yogyakarta, Indonesia. All animals were healthy confirmed with active movements, ate well, hair did not dull or fall out, with bright eyes, and were not blemished. All animals were reared in a controlled temperature environment (25°C±2°C) at a 12-hour dark/light cycle. All efforts were taken to minimize animal suffering during the study: the animal facility was cleaned and disinfected regularly, the animal facility was kept quiet with controlled environmental conditions, and the animals were supplied with c-05 pellets (Citra Ina Feedmill, Jakarta, Indonesia) and water from the national water utility company ad libitum. Before treatment, all rats were acclimatized for seven days.

The animals were divided into five groups, consisting of group K0 (control group), K1, K2, K3 and K4 with arsenic concentration of 5 mg/L, 0.8 mg/L, 0.3 mg/L and 0.01 mg/L, respectively. The control group did not receive any exposure but was placed in the same condition as the treatment groups. Each group (K0-K4) consisted of six rats; during the arsenic exposure process, the animals in each treatment group were placed in separate plastic tub covered with woven iron wire with the same room conditions. All treatment groups were exposed to arsenic over four durations: 2, 4, 6 and 8 weeks (i.e., 14, 28, 42 and 56 consecutive days, respectively).

Ethical statement

The study was conducted from September to December 2020 in the Laboratory of Chemistry/Biochemistry, Faculty of Medicine, Universitas Lambung Mangkurat, Banjarmasin, Indonesia. The protocol of the study was reviewed and approved by the Ethics Committee of the Medical Research, Universitas Lambung Mangkurat (No. 259/KEPK-FK UNLAM/EC/VII/2020, dated 30 July 2020).

Arsenic exposure

Arsenic trioxide with a purity of 99%, purchased from Loba Chemie Laboratory Reagents and Fine Chemicals (Loba Chemie Pvt Ltd, Maharashtra, India), was used to make the arsenic solution. The solution was made by dissolving 5 mg, 0.8 mg, 0.3 mg, and 0.01 mg of arsenic in 1 L of water to produce concentrations of 5 mg/L, 0.8 mg/L, 0.3 mg/L and 0.01 mg/L, respectively. These arsenic levels correspond to the arsenic levels in the geothermal river flow of Ie Seu Um, Aceh Besar District, Aceh Province, Indonesia.³⁰

The arsenic exposure was carried out by immersing the vulva of animals. Soaking was done in a 39 × 42 × 15 cm plastic tub covered with woven iron wire. One soaking tub contained one rat; the immersion limit was as high as the tail until the entire vulva was submerged. The arsenic exposure was conducted over 2, 4, 6, and 8 weeks (i.e., each treatment group had four different exposure times). The exposure was conducted 2.5 h/day. This duration was based on the average length of time that female workers forage for oysters in the estuary of the Ie Seu Um geothermal river.¹³

Uterus homogenate preparation

One day after the end of exposure periods (2, 4, 6 or 8 weeks for K1, K2, K3 and K4, respectively) the animals were euthanized by injecting ketamine 150 mg/kg-xylazine 10 mg/kg intraperitoneally following the guideline of Animal Euthanasia Policy from the Institutional Animal Care and Use Committee.³¹ The uterus was removed during surgery, cut into small pieces and fixed using a phosphate buffer solution with a pH of 7. The small pieces of uterus were mashed to form 5 mL of liquid and centrifuged for 10 min at 3500 rpm.³² The supernatant fraction was collected and used directly to measure the levels of MDA, SOD, H₂O₂, and TNFα.

MDA measurement

A total of 100 μL of uterus homogenate was mixed with 550 μL of distilled water, 100 μL of 10% trichloroacetic acid (TCA), 250 μL of 1 N HCl, and 100 μL of 1% Na-Thio (EMD Millipore Corporation, Billerica, USA). The mixture was then homogenized and centrifuged for 10 min at 500 rpm. Then, the top layer was collected and heated in a water bath at 100°C for 30 min and then cooled at room temperature (25±2°C). The absorbance was measured with a UV–Vis spectrophotometer at a maximum wavelength of 532 nm.³³

SOD measurement

A total of 100 μL uterus homogenate to be measured for SOD levels was incubated for 5 min using 3 mL of 0.05 M Na₂CO₃ and 3 mL of 0.1 M EDTA with a pH of 10.2 and added with 100 μL of adrenaline. The initial absorption was measured (A0) using a spectrophotometer (λ = 480 nm). The solution then incubated again at 30°C for 5 min before determining the final absorption (A1).³⁴

Serum H₂O₂ measurement

A total of 1 mL of uterus homogenate was added to 5 mL of phosphate buffer and slowly homogenized. Then 1 mL of the product of this reaction was taken and 2 mL of dichromate/glacial acetate was added. Heating was done if blue precipitates were formed by heating the tube in a water bath for 10 min until a green color from chromic acetate was formed. The tube was then cooled to room temperature (25 ± 2°C), and H₂O was added to a volume of 3 mL. Next, the solution was placed in a cuvette, and its absorbance was measured by UV–Vis spectrophotometer (λ = 570 nm).^35,36

Measurement of TNFα level

The level of TNFα was measured using the enzyme-linked immunosorbent assay (ELISA) method. The Rat TNFα ELISA kit was used following the manufacture’ protocol (NB-E30635, Novatein Biosciences, Massachusetts, USA). The ELISA was conducted according to the manufacturer’s instructions and the absorbance was read at a wavelength of 450 nm within 30 min.

Uterus histopathology

The uterus tissue was immersed in 10% formalin, cut to a size of 10–15 mm × 10–15 mm × 5 mm, and then proceeded with dehydration, clearing, impregnation, and embedding following standard protocol in Anatomy Pathology.³⁴ Briefly, the tissues were sliced with a thickness of 3–5 μm using a microtome before hematoxylin-eosin (HE) staining. The slices were placed on a slide, immersed in xylol (2 min), absolute ethanol (1 min), and 95% ethanol (1 min) sequentially, then added with water for 10–15 min, and immersed four times in water. The slices were then soaked for 3 min with a hypo solution, rinsed with water for 10 min, then soaked for another 5 min in Mayer’s hematoxylin solution, and watered for approximately 20 min. The preparation was then covered with a coverslip and glued with adhesive.

The uterus histopathology assessment was performed by counting the average number of cells involved in inflammation (macrophages, neutrophils, and lymphocytes). Observations were made using a Meiji Techno microscope with a Sigma HDMI digital photo, 400× magnification. Observation of inflammatory cells was conducted using TopView software through 10 fields of view by paying attention to the histological zone structure of the three preparations found on the slide. The number of inflammatory cells was obtained from the average number of macrophages, neutrophils, and lymphocytes.³⁷ To reduce bias in the study, two pathologists did the observation.

Data analysis

Data were presented as mean ± standard deviation (SD). To compare the levels MDA, SOD, H₂O₂, TNFα and inflammatory cell counts among groups, ANOVA test was used. Duncan’s multiple range tests were used to compare MDA, SOD, H₂O₂, TNFα and inflammatory cell counts between exposure times. All analyses were conducted using a Statistical Analysis System (SAS) version 9.4 (SAS Institute Inc., NC, USA).

MDA levels

Among treatment groups, the highest MDA level was found in the K1 group (5 mg/L) exposed for eight weeks (224.33 ± 1.75 μM) while the lowest concentration was seen for K4 group (0.01 mg/L) exposed for two weeks (174.33 ± 0.51 μM) (Table 1). In the control group, the average MDA levels for the four different exposure times was approximately the same (174 μM). Our data indicated that the higher the arsenic concentration and the longer the exposure time, the higher the uterus MDA levels.

The ANOVA test indicated a significant difference in MDA levels in all groups (p < 0.0001) (Table 1). Duncan’s multiple range tests were used to compare the MDA level between exposure times in each concentration group. The significant difference between exposure time is indicated by different superscript letters (Table 1). There were significant differences in MDA levels between exposure times in all arsenic concentration groups, except for the concentration of 0.01 mg/L exposed for 2 weeks, for whichthere was no difference between control and the 0.01 mg/L group.

SOD levels

The SOD levels in the uterus after exposure to arsenic at different concentrations and exposure times are presented in Table 2. The highest SOD level observed in K4 (0.01 mg/L) group exposed for two weeks while the lowest SOD level was in the K1 (5 mg/L) group exposed for eight weeks. The levels of SOD were significantly different between groups (treatment and control group) with p < 0.0001.

Our data suggest that the longer the exposure time, the lower the levels of SOD. There was a decreasing trend with the length of exposure time. In the K1 group for example, exposure to arsenic at a concentration of 5 mg/L for two weeks produced a SOD of 1.47 ± 0.34 U/mL, and this decreased to 0.36 ± 0.08 U/mL after eight weeks. Duncan’s multiple range test in this group found that there were differences in SOD levels at two, four, six, and eight weeks; however, there was no difference between four and six weeks, nor between six and eight weeks (Table 2).

A similar result was also found in concentrations of 0.8 (K2 group) and 0.3 mg/L (K3 group), for which there was a decrease in SOD levels as the duration of exposure increased (Table 2). The SOD levels were significantly higher for two weeks of exposure compared to the longer exposure time: after the second week, the SOD levels did not change significantly. At the lowest arsenic concentration in accordance with the permissible quality standard concentration (0.01 mg/L), SOD levels in the first two weeks were found at the highest level (2.50 ± 0.46 U/mL) then decreased afterward.

H₂O₂ levels

H₂O₂ is one of the oxidative compounds observed in this study, and the levels of uterus H₂O₂ after exposure to arsenic at various concentrations and durations are presented in Table 3. Our data suggested an increase in H₂O₂ levels at each additional exposure time in every arsenic concentration group. The mean H₂O₂ level in the control group after eight weeks was 2.33 ± 0.07 μM. This figure was significantly different from the other treatment groups (p < 0.0001). The multivariate test also showed a significant difference in the mean H₂O₂ levels between each treatment group (Table 3).

TNFα levels

Table 4 presents the TNFα levels after exposure to arsenic in all treatment groups. Drastic spikes in all concentration groups were observed in the six- and eight-week exposure groups. These data suggest that the uterus inflammatory response to arsenic exposure through the vulva increased rapidly when arsenic exposure lasted more than 4 weeks. Overall, there were significant differences between the treatment and control groups (p < 0.0001). Post-hoc tests in all concentration groups also showed significant difference between the two- and four-week groups and the six- and eight-week groups (Table 4).

Uterus histopathology

The uterus tissues from all animals were stained with HE and observed with 400×magnification. The total number of inflammatory cells in each group and statistical analysis test results are presented in Table 5. There was a significant difference of the number of inflammatory cells in arsenic exposure between length of exposures in all concentration groups (p < 0.0001). A further test using Duncan’s multiple range test found that there was no difference in inflammation at 5 mg/L arsenic exposure between two, four, and six weeks. The difference was only seen after eight weeks exposure. The arsenic concentration of 0.8 mg/L exposed through the vulva did not significantly differ between two, four, six, and eight weeks of treatment duration. At a concentration of 0.3 mg/L, there was a difference between two and four weeks of exposure and six and eight weeks of exposure. The 0.01 mg/L concentration group did not show any significant differences in any exposure time.

H&E staining on the uterus tissue suggested that inflammation occurred in the lining of the endometrium, myometrium, and perimetrium (Figure 1). Inflammatory cells were also found in the uterus fatty tissue. The representation of the histopathology of inflammatory cells in the uterus lining from all groups are presented in Figure 1.

Figure 1. Histopathology of inflammatory cells in the uterus lining of Rattus norvegicus exposed to 5, 0.8, 0.3 and 0.01 mg/L arsenic through the vulva.

(A) infiltration of inflammatory cells occurred in all treatment groups. Inflammatory cells were found in the endometrium, myometrium, and perimetrium and reached the fat tissue. (B) Infiltration of N, M, and L occurred in all treatment groups (2, 4, 6, and 8 weeks). The inflammatory cells reached the myometrium layer, perimetrium layer, and fat tissue. (C) Infiltration of inflammatory cells in the control group was dominated by N. In the treatment group, the inflammatory cell infiltration contained N and M and also L. Inflammatory cells occurred in the lining of the endometrium, myometrium, and perimetrium. (D) Infiltration of inflammatory cells was found in all treatment and control groups. Most M and L were found in the treatment at 4 and 8 weeks. The inflammatory cells also reached the perimetrial lining. N: neutrophils, M: macrophages, L: lymphocytes.