F1000Research F1000Research 2046-1402 F1000 Research Limited London, UK 10.12688/f1000research.129833.1 Research Article Articles Effect of arsenic exposure on MDA, SOD, H 2O 2 and TNF-α levels of uterus homogenate of female Sparague-Dawleys rats

[version 1; peer review: 1 approved, 1 approved with reservations]

 Irnawati Irnawati Conceptualization Formal Analysis Funding Acquisition Methodology Project Administration Resources Supervision Writing – Original Draft Preparation https://orcid.org/0000-0003-4950-5504 a 1 Idroes Rinaldi Data Curation Methodology Resources Software Validation Writing – Review & Editing 2 3 Akmal Muslim Resources Validation Visualization Writing – Review & Editing 4 Suhartono Eko Resources Supervision Validation Writing – Review & Editing 5 Seriana Irma Resources Validation Visualization Writing – Review & Editing 1 Department of Midwifery, Aceh Polytechnic of Health, Aceh Besar, 23241, Indonesia Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Syiah Kuala, Banda Aceh, 23111, Indonesia Department of Pharmacy, Faculty of Mathematics and Natural Sciences, Universitas Syiah Kuala, Banda Aceh, 23111, Indonesia Laboratory of Histology, Faculty of Veterinary Medicine, Universitas Syiah Kuala, Banda Aceh, 23111, Indonesia Department of Medical Chemistry/Biochemistry, Faculty of Medicine, Lambung Mangkurat University, Banjarbaru, Kalimantan Selatan, 70124, Indonesia irnawati@poltekkesaceh.ac.id

No competing interests were disclosed.

 30 1 2023 2023 12 108 12 1 2023 Copyright: © 2023 Irnawati I et al. 2023 This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Background and Aim: Arsenic exposure to the body through the oral, dermal, and inhalation routes have a detrimental impact on health, including women’s reproductive health. However, the effect of arsenic exposure through the vulva of women is unclear. The present study therefore examined the effects of long-term arsenic exposure of vulva on uterus inflammation mediated by oxidative stress and inflammatory mechanism.

Materials and Methods: Female Rattus norvegicus L was used as the animal model and the arsenic were exposed through vulvar immersion. The arsenic solution was made into four concentrations while the duration of exposure was made in four-time combinations. Uterus inflammation was assessed through histopathological observation of uterus tissue through hematoxylin and eosin (H&E) staining. Oxidative stress was assessed using malondialdehyde (MDA), superoxide dismutase (SOD), and hydrogen peroxide (H 2O 2); while inflammatory profile was assessed by measuring using tumor necrosis factor α (TNFα).

Results: Our data suggested that inflammation could occur at the permissible quality standard concentrations when arsenic was exposed for more than two weeks. At higher concentrations and a longer exposure time, arsenic exposure could lead to chronic inflammation. Arsenic exposure was able to increase the levels of MDA and H 2O 2 and reduced the SOD suggesting stress oxidative of the organ. Arsenic also could increase the level of TNFα at any concentration after 6 and 8 weeks of exposure suggesting the inflammation process in the uterus.

Conclusion: Continuous exposure of vulva with arsenic could induce oxidative stress and chronic inflammation of the uterus. Further study to investigate this finding in human is critical as basic to propose health campaign program to the community in the high arsenic regions.

 Arsenic exposure uterus inflammatory vulva absorption vulva exposure The author(s) declared that no grants were involved in supporting this work. Introduction

Heavy metal exposure is still often found in humans. 1 Heavy metals in the environment come not from industrial pollution as well as nature itself. 2 Hot springs, 3 , 4 water blowouts, 5 volcanic eruptions, and abrasion are examples of the natural activities that produce and supply the heavy metal into environment. 6 This makes heavy metal exposure to the organisms difficult to avoid. Therefore, assessing potential pollution, heavy metal exposure mechanisms, and their health effects are important. 7 The effects of heavy metal exposure on an organism are significant, since it could easily interact with many organs. 8 , 9 Frequent exposure can also deposit the metal inside the body. 10 Although the organism has its own outer protective barrier, 11 , 12 it still has some open organs, which can be the heavy metals’ point of entry into the body. 13

The female external genital organs are open 14 and they could become the entrance for toxic agents into the body. The tissue structure of the vulva and vagina is more permeable than the skin because vulvar tissue has the ability to hydrate and reduce water barrier function. 15 In addition, most of the vaginal wall is covered by blood and lymphatic vessels, and therefore chemical exposure might be directly absorbed into the circulatory system. 16 It is also very easy for the vulva and vagina to absorb toxic chemicals, and absorbed chemicals can be easily and effectively distributed through the body. 17

The entry of harmful chemicals into the body through the female reproductive organs can be caused by many activities. Most are linked to the use of feminine hygiene or feminine care products. 18 These various products are used by attaching them to the vulva, smearing, douching, and evaporating into the vagina or through immersion. The basic ingredients of these products can contain dioxin, chlorine, pesticide residues, methyldibromo glutaronitrile, methylchloroisothiazolinone, methylisothiazolinone, parabens, quaternium-15, DMDM hydantoin, and other hazardous substances. 17 Apart from these various products, other activities that allow the entry of harmful chemicals into the body through the genital organs can occur while bathing, wiping, swimming, and soaking.

Women in Aceh Province, Indonesia use river estuaries for their daily livelihoods. They collect various estuarine biota such as oysters and shellfish at the bottom of the estuary by sitting or squatting in the estuary. During work, estuary water containing hazardous substances wet the genital organs. A study found that the work is performed for 2–3 h/day without using personal protective equipment. 13 The north zone of the geothermal area around Seulawah Agam volcano of Aceh Province has a hot spring feature. 19 , 20 This hot water flows and joins the nearest river 21 such as Ie Seu Um geothermal river that crosses a residential area in Mesjid Raya Sub-District, in the Aceh Besar District. 22 This river contains various levels of arsenic: the highest level found in water sources was 5478 μg/L (5 mg/L); the arsenic level in local wells was 800 μg/L (0.6 mg/L), and those at river estuaries was 300 μg/L (0.3 mg/L). 23 Meanwhile, the quality standard concentration for arsenic level in water set by the World Health Organization (WHO) and the Ministry of Health of the Republic of Indonesia is only 10 μg/L (0.01 mg/L). 24 , 25

The effects of arsenic on the female reproductive system not only cause toxicity to the reproductive organs, decreases libido, and teratogenic disorders, but it could also affect the well-being of a fetus, for example through congenital malformation. 26 – 28 Arsenic exposure to the body causes metabolic toxicity in cells as well as oxidative stress and as a result, causes lipid peroxidation, membrane damage, membrane peroxidation and destruction, which might lead to cell death. 29 Many studies assessed the effects of arsenic exposure through oral, ingestion, inhalation, parenteral, and dermal on damages to the reproductive system. However, research assessing the effect of arsenic exposure through the vulva and vagina on uterus damages is limited.

The present study aimed to assess the effects and damages of arsenic exposure through the vulva on the uterus using animal model . The concentration was adjusted to the arsenic levels in the geothermal area of Ie Seu Um, Aceh Besar Regency (5, 0.8, 0.3, and 0.01 mg/L) and the exposure was applied for 2, 4, 6, and 8 weeks. The oxidative effect was examined through the levels of malondialdehyde (MDA), superoxide dismutase (SOD), and hydrogen peroxide (H 2O 2) of the uterus tissue. Uterus inflammation was studied by measuring the level of tumor necrosis factor α (TNFα) and examining the histopathology of the uterine tissue.

 Methods 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, H 2O 2, 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 Na 2CO 3 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 H <sub>2</sub>O <sub>2</sub> 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 H 2O 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, H 2O 2, TNFα and inflammatory cell counts among groups, ANOVA test was used. Duncan’s multiple range tests were used to compare MDA, SOD, H 2O 2, 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).

 Results 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.

Comparison of MDA levels of uterus post exposure to arsenic at different concentrations.
Groups MDA levels (mean ± SD, μM) p-value
2 weeks 4 weeks 6 weeks 8 weeks
K0 (control) 174.17 ± 0.40 m 174.33 ± 0.51 m 174.50 ± 0.54 m 174.33 ± 0.48 m <0.0001
K1 (5 mg/L) 182.52 ± 4.23 ji 189.67 ± 0.81 h 206.50 ± 0.54 d 224.33 ± 1.75 a
K2 (0.8 mg/L) 181.60 ± 0.54 jk 183.67 ± 1.03i 202.17 ± 0.98 e 218.00 ± 3.03 b
K3 (0.3 mg/L) 179.80 ± 0.89 k 182.00 ± 0.89 ji 200.33 ± 0.51 f 210.17 ± 1.72 c
K4 (0.01 mg/L) 174.33 ± 0.51 m 177.67 ± 1.72 l 194.17 ± 0.75 g 199.50 ± 1.04 f

Note: Means with the same letter are not significantly different p ≤ 0.05.

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.

Comparison of SOD levels of uterus post exposure to arsenic at different concentrations.
Groups SOD level (mean ± SD, U/mL) p-value
2 weeks 4 weeks 6 weeks 8 weeks
K0 (control) 0.91 ± 0.05 def 0.96 ± 0.19 def 0.95 ± 0.09 def 0.95 ± 0.07 def <0.0001
K1 (5 mg/L) 1.47 ± 0.34 c 0.68 ± 0.08 fgh 0.51 ± 0.06 ih 0.36 ± 0.08 i
K2 (0.8 mg/L) 1.68 ± 0.32 c 0.86 ± 0.20 defg 0.79 ± 0.04 defg 0.58 ± 0.08 hig
K3 (0.3 mg/L) 2.03 ± 0.27 b 0.98 ± 0.08 de 0.87 ± 0.06 defg 0.74 ± 0.07 efgh
K4 (0.01 mg/L) 2.50 ± 0.46 a 1.49 ± 0.50 c 1.09 ± 0.17 d 1.06 ± 0.18 d

Note: Means with the same letter are not significantly different p ≤ 0.05.

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.

 H <sub>2</sub>O <sub>2</sub> levels

H 2O 2 is one of the oxidative compounds observed in this study, and the levels of uterus H 2O 2 after exposure to arsenic at various concentrations and durations are presented in Table 3. Our data suggested an increase in H 2O 2 levels at each additional exposure time in every arsenic concentration group. The mean H 2O 2 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 H 2O 2 levels between each treatment group ( Table 3).

Comparison of H <sub>2</sub>O <sub>2</sub> levels of uterus post exposure to arsenic at different concentrations.
Groups H 2O 2 levels (mean ± SD, μM) p-value
2 weeks 4 weeks 6 weeks 8 weeks
K0 (control) 2.32 ± 0.06 k 2.35 ± 0.08 k 2.29 ± 0.08 k 2.33 ± 0.07 k <0.0001
K1 (5 mg/L) 5.42 ± 0.54 h 7.05 ± 0.41 cd 7.66 ± 0.09 b 8.12 ± 0.18 a
K2 (0.8 mg/L) 4.89 ± 0.42 i 6.85 ± 0.31 d 7.38 ± 0.04 cb 7.64 ± 0.08 b
K3 (0.3 mg/L) 4.60 ± 0.29 i 6.01 ± 0.04 g 6.51 ± 0.09 e 6.89 ± 0.05 d
K4 (0.01 mg/L) 3.68 ± 0.78 j 5.56 ± 0.13 h 6.18 ± 0.05 f 6.47 ± 0.14f e

Note: Means with the same letter are not significantly different p ≤ 0.05.

 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).

Comparison of TNFα of uterus post exposure to arsenic at different concentrations.
Groups TNFα levels (pg/mL) p-value
2 weeks 4 weeks 6 weeks 8 weeks
K0 (control) 279.60 ± 16.71 j 275.27 ± 16.19 j 268.24 ± 8.27 j 277.53 ± 2.69 j <0.0001
K1 (5 mg/L) 355.38 ± 25.16 hi 369.32 ± 26.69 h 1702.31 ± 87.72 c 2129.82 ± 80.71 a
K2 (0.8 mg/L) 323.80 ± 1.56 hij 339.41 ± 28.91 hi 1408.05 ± 76.97 d 1836.39 ± 45.90 b
K3 (0.3 mg/L) 313.47 ± 2.58 hij 319.60 ± 3.53 hij 1252.21 ± 28.63 e 1396.83 ± 64.31 c
K4 (0.01 mg/L) 306.12 ± 3.60 ij 315.31 ± 10.31 hij 1063.27 ± 45.74 g 1149.90 ± 31.19 f

Note: Means with the same letter are not significantly different p ≤ 0.05.

 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.

Comparison inflammation cell count of uterus post exposure to arsenic at different concentrations.
Groups Inflammation cells (mean ± SD) p-value
2 weeks 4 weeks 6 weeks 8 weeks
K0 (Control) 8.67 ± 1.21 e 8.67 ± 1.21 e 8.67 ± 1.21 e 8.67 ± 1.21 e <0.0001
K1 (5 mg/L) 26.37 ± 10.88 a 23.37 ± 13.54 ab 21.83 ± 14.55 abc 19.83 ± 3.94 cde
K2 (0.8 mg/L) 18.53 ± 2.91 abcde 23.27 ± 5.70 ab 18.37 ± 11.02 abcde 15.93 ± 6.46 abcde
K3 (0.3 mg/L) 24.50 ± 11.60 a 26.77 ± 9.04 a 10.43 ± 2.65 de 19.67 ± 9.20 abcde
K4 (0.01 mg/L) 19.87 ± 10.14 abcd 18.57 ± 8.56 abcde 19.70 ± 8.35 abcde 12.83 ± 3.31 bcde

Note: Means with the same letter are not significantly different p ≤ 0.05.

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.

Histopathology of inflammatory cells in the uterus lining of <italic toggle="yes">Rattus norvegicus</italic> 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.

 Discussion

Once absorbed by the vulva and vagina, arsenic seemed to increase its oxidative effect in the uterus. This is supported by increasing MDA and H 2O 2 levels and decreasing SOD levels in the uterus of experimental animals. Arsenic exposure rapidly stimulates the formation of intracellular reactive oxygen species (ROS). The ROS formed causes changes in how cells work. The contaminated cells might carry epigenetic modifications and changes in signaling pathways. ROS are even capable of causing direct oxidative damage to molecules. The induction of ROS production by arsenic in cells occurs via the mitochondrial electron transport chain and exerts a toxic effect on the mitochondria by inhibiting the activity of succinate dehydrogenase and releasing the oxidative phosphorylation pair through the production of superoxide anions to form other ROS. 38 , 39 Another mechanism occurs through the deployment of nicotinamide adenine dinucleotide phosphate, which is oxidized (Nox). Nox is a membrane enzyme that produces ROS as a result of arsenic exposure. Nox accelerates the formation of superoxide anions. Another factor is that the metabolism of arsenic itself triggers the formation of ROS in cells, such as singlet oxygen, H 2O 2, superoxide anions, and SOD. 38 , 39

Our data also suggested that the arsenic exposure at all concentrations induced the uterus MDA levels from a two-week exposure except for the 0.01 mg/L concentration. At this standard concentration, when exposed for two weeks, it did not cause oxidative effects. However, when the exposure lasted more than two weeks, the oxidative effect could still be observed. Arsenic exposure caused oxidative stress to the uterus in experimental animals after two weeks, indicated by the oxidative biomarker of MDA. MDA is a marker of oxidative stress derived from the end product of lipid peroxidation produced by free radicals. 40 Our data suggests that the longer the exposure time and the higher arsenic concentration, the higher the ROS levels.

Slightly different from MDA, arsenic exposure induced H 2O 2 production even for the lowest concentration (0.01 mg/L). The permissible quality standard concentration of arsenic could increase the H 2O 2 level after two weeks exposure. The results also showed a decrease in SOD levels. SOD is an antioxidant defense enzyme, being the first line of defense against the occurrence of OS. SOD also acts as an enzyme detoxifying ROS and eliminating free radicals. 41 , 42 Our data suggested arsenic exposure increased ROS and decrease the level of SOD. This condition could increase lipid peroxidation, endometrial cell growth and adhesion, and increase the formation of macrophages leading to an imbalance between antioxidants, especially SOD and ROS. 43 This imbalance disrupts angiogenesis and blood supply, and causes persistent inflammation. 43

Our present study also found that arsenic at the quality standard limit, when exposed continuously for more than two weeks, could cause oxidative effects. This caused the emergence of more ROS in the uterus, leading to infiltration of more macrophages. The higher the arsenic level and the longer the exposure, the higher the number of macrophages in the uterus tissues. Macrophages are part of the innate immune system, coordinate the process of the adaptive immune responses, restoring tissue homeostasis, inflammatory processes, and repair activities, and are also considered to be pro-inflammatory immune cells. 44 The presence of macrophages could activate TNFα, a pro-inflammatory agent capable of regulating many aspects of macrophage function. TNFα can be released rapidly following toxicity, infection, or exposure to lipopolysaccharides and is considered to be a major regulator of pro-inflammatory cytokine production. Apart from pro-inflammatory cytokines, TNFα also enhances lipid signal transduction mediators such as prostaglandins and platelet-activating factors. 45 Therefore, TNFα is considered to be the main cause of the activation and recruitment of inflammatory cells and plays an important role in chronic inflammation. 45 Arsenic induces ROS production by changing antioxidant enzymes such as catalase and SOD to trigger the appearance of lymphocytes in inflamed tissues. 46

Inflammation is a protective response that aims to protect tissues from becoming necrotic, eliminate the causes of infection, and stimulate tissue healing and repair processes. When inflammation occurs, neutrophils will appear as the body’s first defense cells against injury or infection. Macrophages will help the process of eliminating infection and damaged tissue through the process of phagocytosis. Lymphocytes play a role in chronic inflammation. 47 , 48 Chronic inflammation occurred in the arsenic exposure groups with 0.5, 0.8, and 0.3 mg/L concentrations. This chronic inflammation was characterized by the appearance of abundant lymphocytes. The appearance of this accumulation of leukocytes is due to tissue injury resulting from a long-term inflammatory response. 49 Although macrophages and lymphocytes were found in animal exposed to 0.01 mg/L arsenic, the number was significancy smaller. However, exposure for more than two weeks also could present inflammation responses. Our data also found that neutrophils were present in the uterus in all treatment groups. Inflammatory cells were also present in the control group in very small numbers and only neutrophils and macrophages were present. Neutrophils are the first cells present when there is tissue injury and infection and are associated with the body’s defense against infection and other inflammatory processes and it is also known as the first responder and constant defender. 47

Conclusions

Our data suggest that continuous and long-term immersion of animal vulva with water containing arsenic even at the permissible concentration (0.01 mg/L) could cause inflammation of the uterus tissues. Arsenic at 0.3, 0.8, and 5 mg/L could cause chronic inflammation of the uterus after a two-week exposure. Arsenic exposure was able to increase the uterus MDA levels even at 0.01 mg/L when exposed for more than two weeks. Arsenic increased the levels of H 2O 2 at all concentrations after a two-week exposure. In contrast, arsenic also induced the ROS by reducing the levels of SOD in uterus. Arsenic could increase the level of TNFα at any concentration after six and eight weeks of exposure.

 Data availability Underlying data

Figshare: ‘Damage to the uterus due to arsenic exposure to the vulva via oxidative stress (MDA, SOD and H 2O 2) and inflammatory (TNF-α) pathways of female Sprague-Dawley rats’. DOI: https://doi.org/10.6084/m9.figshare.21774989. 50

This project contains the following underlying data:

Master Table.xlsx [Table containing the raw data of the study].

 Reporting guidelines

Figshare: ARRIVE checklist for ‘Damage to the uterus due to vulva exposure to arsenic via oxidative stress (MDA, SOD and H 2O 2) and inflammatory (TNF-α) pathways of female Sprague-Dawley rats’. https://doi.org/10.6084/m9.figshare.21774989. 50

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

 Acknowledgments

Authors would like to thank the Indonesian Ministry of Health for providing the fund for this study.

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Journal Contribution. 2022. 10.6084/m9.figshare.21774989.v2 10.5256/f1000research.142546.r183681 Reviewer response for version 1 Ahmad Aqeel 1 Referee University of Chinese Academy of Science, Beijing, China

Competing interests: No competing interests were disclosed.

 25 8 2023 Copyright: © 2023 Ahmad A 2023 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve-with-reservations

General evaluation: The content of the manuscript is interesting and important for a wide range of readership in this research field. The article is well organized. The abstract of the paper is compressed, realistic, and understandable.

The manuscript contains original data. The innovation of the paper is strongly connected to the journal. The introduction and discussion sections look good, summarize the current state of the problem with the elaboration of the theme, and provide scientific key information about the work. The introduction should briefly show what is already known and then focus more on what is not known and why it is worth studying. The philosophy of this work is good, and applicable in the next research.

Please include the missing information (research gaps and the significance of your research). Why is it required to run such an investigation?

Title: It is realistic and attractive.

Abstract: Abstract is integral and can serve as a stand-alone document that succinctly describes both procedures and conclusions.

Introduction: The literary structure of the Introduction section is good, with a simple but scientific objective, containing essential information about the work and critical information on the problem under study.

The presentation of the results in the form of graphs and tables is clear, and the results are well described. All results are comprehensive. However, the Discussion section could be improved to make the manuscript more attractive. I have made a suggestion about the Discussion section. But overall, the discussion is sufficient.

The conclusions are well formulated and justified. The research meets all applicable standards for research integrity. The presentation of the results and figures facilitated the understanding of the experimental results. A deeper mechanistic understanding of the measured parameters is missing.

References - include more recent references throughout the manuscript, specifically in the discussion, to increase the value of the paper. I don’t put much stress at this point, but in my suggestion, some new references can be added to show the complexity of the topic.

Final comments: The manuscript is interesting, associated with actual plant science trends. My opinion is in support of the manuscript to be indexed.

I propose acceptance of this manuscript, with recommended minor revisions.

Is the work clearly and accurately presented and does it cite the current literature?

Yes

If applicable, is the statistical analysis and its interpretation appropriate?

Yes

Are all the source data underlying the results available to ensure full reproducibility?

Yes

Is the study design appropriate and is the work technically sound?

Yes

Are the conclusions drawn adequately supported by the results?

Yes

Are sufficient details of methods and analysis provided to allow replication by others?

Yes

Reviewer Expertise:

Life sciences

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

 10.5256/f1000research.142546.r162541 Reviewer response for version 1 Michalak Izabela 1 Referee https://orcid.org/0000-0001-8084-9642 Department of Advanced Material Technologies, Faculty of Chemistry, Wroclaw University of Science and Technology, Wroclaw, Poland

Competing interests: No competing interests were disclosed.

 14 2 2023 Copyright: © 2023 Michalak I 2023 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve

The article raises the important issue of women's exposure to arsenic. The results obtained in this publication may be helpful in raising public awareness of the dangers of staying in water contaminated with arsenic or other heavy metals.

Generally this manuscript is well written. In my opinion, manuscript in the present form can be accepted after minor revision.

Some specific comments on this paper:

Abstract:

Materials and Methods: should be “Female rat Rattus norvegicus L .”

Not arsenic was exposed “the arsenic were exposed through vulvar immersion” but for example “exposure to arsenic occurred by immersing the vulva in water solutions of this heavy metal”

“…into four concentrations…” – I think it will be beneficial to add here in the bracket these concentrations

“…made in four-time combinations” – I think it will be beneficial to add here in the bracket these exposure periods

“…At higher concentrations and a longer exposure time…” – please indicate these results

Change the order of Keywords and add “rat”: Rat, arsenic exposure, vulva exposure, vulva absorption, uterus inflammatory. Keywords should be arranged in a logical manner, in the order in which the research is conducted

 Introduction:

“The basic ingredients of these products can contain dioxin…” – it looks like all hygiene products are very polluted/contaminated. I would advise caution. What's more, this data comes from a 2013 report published a decade ago. It's better to cite a current source

 Methods:

“…made by dissolving 5 mg, 0.8 mg, 0.3 mg, and 0.01 mg of arsenic in 1 L” – it is not clear for me. Did you dissolve in water salt – arsenic trioxide? Was the concentration calculated to arsenic alone or to an arsenic salt? Then we have two different output concentrations

“..measure the levels of MDA, SOD, H 2O 2, and TNFα” – should be: TNF-α – please standardize in the whole manuscript

“..10% trichloroacetic acid” – please add the name of the Producer, city and country for all chemicals you used in this study

how was the MDA, SOD, H 2O 2, TNF-α level calculated from absorbance? This should be added to the manuscript

a model of UV–Vis spectrophotometer (Producer) should be added

Data analysis: you present your data as a mean and standard deviation. Did you check normality of distribution of your results? Only for normal distribution we can apply mean

 Results:

here, before the section “MDA levels”, it is worth adding an introductory sentence

unify the number of significant digits in all tables

H 2O 2 levels and TNF-α level: lack of a more detailed interpretation of the results, such as for MDA and SOD level – please add

In Table 2: SOD level s

Figure 1 – the inscriptions in the picture are very hard to see, please increase the font size and its color

 Discussion:

Should be “…and decrease d the level of SOD”

“by changing antioxidant enzymes such as catalase and SOD to trigger…” – why the level of catalase was not studied in this study?

 Conclusions:

conclusions should be supported by research results

in the future, it is worth examining the accumulation of arsenic in the examined tissues

Is the work clearly and accurately presented and does it cite the current literature?

Yes

If applicable, is the statistical analysis and its interpretation appropriate?

Yes

Are all the source data underlying the results available to ensure full reproducibility?

Partly

Is the study design appropriate and is the work technically sound?

Yes

Are the conclusions drawn adequately supported by the results?

Partly

Are sufficient details of methods and analysis provided to allow replication by others?

Yes

Reviewer Expertise:

Environment pollution; heavy metal ions; Biomonitoring; Multielemental analysis of biological samples

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.