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International Journal of Experimental Pathology logoLink to International Journal of Experimental Pathology
. 2015 Apr 17;96(2):111–115. doi: 10.1111/iep.12123

Application of talcum powder, trichloroacetic acid and silver nitrate in female rats for non-surgical sterilization: evaluation of the apoptotic pathway mRNA and miRNA genes

Onder Yumrutas *, Murat Kara †, Remzi Atilgan ‡, Salih Burcin Kavak ‡, Ibrahim Bozgeyik *,✉, Ekrem Sapmaz ‡
PMCID: PMC4459804  PMID: 25885949

Abstract

There are several methods used for non-surgical sterilization in birth control including quinacrine, trichloroacetic acid (TCA), erythromycin, tetracycline, silver nitrate and talcum powder. Among these, talcum powder, TCA and silver nitrate are the most commonly used. However, the toxic and carcinogenic activities of these chemicals in ovarian tissue have been poorly elucidated. This study demonstrates the expression levels of antioxidant, apoptotic and anti-apoptotic genes after administration of talc powder, TCA and silver nitrate for non-surgical sterilization in female rat models. The expression changes of some microRNAs (miR-15b, miR-21, miR-34a and miR-98) that play key roles in the apoptosis pathway were also included. All expression analyses were evaluated with real-time PCR. The expression levels of all genes appeared to be upregulated in the talcum powder group, but the results were not statistically significant. Increased expression of Gsr and Sod1 genes was statistically significant in the talcum powder group. In TCA and silver nitrate group, expression of all genes was appeared to be elevated but only the Gsr expression was statistically significant in the TCA-administrated group; there were no statistically significant changes in the silver nitrate group. miRNA expression levels were increased in talcum powder and TCA-administrated groups, but these results were not significant. Expression levels of miR-15b, miR-21 and miR-98 in the silver nitrate group were significantly increased. Consequently, these chemicals appear to be non-carcinogenic agents for rat ovarian tissue which do not induce apoptosis. However, talcum powder and TCA can be considered as agents that are toxic to ovarian tissue.

Keywords: antioxidant, apoptosis, miRNA, silver nitrate, talcum powder, trichloroacetic acid

Non-surgical sterilization is one of the most widely used of birth control methods. Many types of non-surgical sterilization methods are used for this purpose. Quinacrine is the most commonly used and is the method of choice (Benagiano 2001; Kara et al. 2014). In addition to quinacrine, trichloroacetic acid (TCA), erythromycin, tetracycline, silver nitrate and talcum powder have been used in non-surgical sterilization. However, there have been only limited number of studies focused on the effects of chemicals that are used in non-surgical sterilization on cells and tissues at molecular level.

Moreover, microRNAs are important regulators of gene expression at post-transcriptional level (Bartel, 2004, Calin and Croce, 2006). They function through binding 3'-UTRs of target mRNAs, thus mediating either degradation target mRNA or translation inhibition of the target mRNA (Bartel, 2004). Also, they have been shown to participate in a variety of important biological pathways including proliferation, differentiation, apoptosis, and inflammation. They are small in size, approximately 22 nucleotides long, and regulate gene expression at post-transcriptional level. Several studies have reported that miRNAs have prognostic, diagnostic and therapeutic potential. Therefore, miRNA expression data can give useful information that can have wide implications.

In our previous study we investigated the effects of quinacrine, tetracycline and erythromycin in non-surgical sterilization by exploring their effects on the expression of important antioxidants and apoptotic genes as well as related microRNAs in rat ovarian tissue (Kara et al. 2014). Our previous results have shown that those substances, that are all used in non-surgical sterilization, have no effect on the expression of antioxidant and apoptotic associated genes. However, it is important to determine the toxic effects of the other agents, such as talcum powder, trichloroacetic acid (TCA) and silver nitrate, that are also used in non-surgical sterilization, on tissues. TCA, when appliedtopically, denatures proteins and causes chemical cauterization. It is frequently used in treatment of genital papillomata. When TCA was used for endometrial ablation it caused widespread atrophy of the endometrium with adhesions and formation of fibrous bands. The use of TCA in endometrial ablation is effective, well tolerated, easily implemented, cheaper and does not need hospitalization for the treatment of functional menorrhagia (Kucukozkan et al. 2004). In addition the carcinogenic properties of TCA have been reported in the literature (Pereira & Phelps 1996). In particular, TCA increases the proliferation of cancer cells (Ge et al. 2001). Additionally, silver nitrate has cytotoxic effects especially on target cells that are sensitive to tissue regeneration. It enables the breakdown of proteins in cells and thus inhibits proliferation and differentiation in a variety of cells (Hidalgo & Dominguez 1998). Talcum powder causes a chronic granulomatous reaction with the formation of fibrosis and adhesions (Cox et al. 1996). It is used as adhesion stimulating treatment in both benign and neoplastic pleural effusions and effective results have been obtained (Tampinco et al. 1995). In addition, talcum powder and ovarian cancer have been linked, but there is no definite evidence to support the hypothesis that talcum powder increases risk of developing cancer (Whysner & Mohan 2000). In several studies, talcum powder has shown a positive response to carcinogenic agents (Hamilton et al. 1984). Yet, studies concerning the effect of talcum powder have been reported to be controversial and insignificant and further epidemiological and experimental studies are needed. Inflammation is one of the hallmarks of cancer, and talcum powder is known to induce inflammation in tissues (Froudarakis et al. 2006). Thus, a clear understanding is needed in terms of inflammation induced by the talcum powder. Laparoscopic sterilization is a popular methods of choice in family planning in some parts of the world today.

Thus it is important to determine the expression levels of molecular changes in order to indicate what may occur after laparoscopic sterilization. Consequently, the aim of this study was to show the effects of the talcum powder, TCA and silver nitrate on rat ovarian tissue and to evaluate the expression levels of related genes at mRNA level and apoptotic miRNAs genes.

Materials and methods

The study was carried out at the Experimental Medical Genetics Laboratories, Research Centre, of Firat University. A total of 28 Wistar albino female rats were used. Rats were 12–14 week old with regular cycles and 200–220 g in weight. The study was approved by the Institutional Ethics Committee of Firat University with the accesion number: 16.02.2012/25.

For the regular biological rhythms, rats were divided into four groups and retained in separate cages with the following conditions: 12 hours artificial light, 12 hours darkness and 21–23°C. Rats were fed with standard pellet feed and tap water (Principles of Laboratory Animal Care; NIH publication no: 86-23, revised 1985). Oral feeding of animals was ended 18 h prior to the experiment and was allowed to drink tap water only. Animals were divided into four random, prospective, single-blind groups as presented in Table1.

Table 1.

Experimental groups and applied protocol

Groups Applications
Group 1 (n = 7) Control group, no application performed
Group 2 (n = 7) 1 cm linear incision made in right uterine horn distal segment, intraluminal 2 × 2 mm (100 mg/kg) talcum powder was inserted and the incision closed with 4/0 vicryl
Group 3 (n = 7) % 100 TCA was applied transcervically to theendometrial surface for one minute
Group 4 (n = 7) % 100 silver nitrate was applied transcervically to endometrial surface for one minute
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The study substances were applied during the proliferative phase of the menstrual cycle of rats. The study lasted for one month. Applications and amount of substances used are shown in Table1. The ovarian tissues were excised at the end of the month. Temperature, blood pressure and heart rate of the animals was monitored continuously during substance applications. All parameters were normal, and no abnormal situation was observed. The ovarian tissue was excised at the end of the experiment. Subsequently, ovarian tissue was stored at −80°C for the total RNA isolations.

Total RNA extraction, cDNA synthesis and real-time PCR analysis

Total RNA of the overian tissues was isolated using a QIAamp RNA isolation kit (QIAGEN Sample & Assay Technologies, Hilden, Germany) with additional DNase treatment step. RNA samples were kept at −80°C until expression analysis. For the miRNA isolations, miRNeasy Mini Kit from Qiagen were used and manufacturer's recommended protocol was followed. For the preparations of cDNA samples, Qiagen miScript Reverse Transcription Kit (Qiagen Sample and Assay Technologies) was used. Gene expression levels were assessed by real-time PCR (RT-PCR) method. For the real-time PCR analysis, primers of the selected mRNAs were designed using ‘Gene’ interface of NCBI database (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Primers for the mature miRNAs were purchased from Qiagen, and U6 small RNA was used as an internal control for the normalization of miRNA expression data. For the gene expression studies, SYBR Green Master Mix (Thermo Fisher Scientific Inc., Waltham, MA, USA) was used. Gapdh gene was used as internal control to normalize mRNA expression data. Real-time quantifications of transcripts were held in Rotor Gene 6000 Real-Time PCR Machine.

Statistical analysis

qPCR measurements were calculated by the formula of 2−ddCT. The statistical analysis was conducted using spss package program (IBM Corporation, New York, USA) for Windows. In the statistical analysis of data, Kruskal–Wallis variance analysis and Mann–Whitney U-test were used. All statistical analysis was two-tailed, and P < 0.05 were accepted as significant.

Results

Determination of mRNA and miRNA expressions levels by real-time PCR method

To evaluate the mRNA and miRNA expression levels of selected genes in rat ovarian tissue, real-time PCR method was used. Expression levels of antioxidant, apoptotic and anti-apoptotic genes were presented in Table2. Expression levels in experimental groups were compared to control group and statistically analysed. In the group which had talcum powder administered group, the expression levels of most genes were upregulated, but the results were not statistically significant. The increased expression of Gsr and Sod1 genes was statistically significant. In the TCA and silver nitrate group, mRNA levels of all genes were determined to be increased. However, only the Gsr expression in the group to which TCA had been administered was statistically significant.

Table 2.

mRNA expression levels of selected antioxidant, antiapoptotic and apoptotic genes

Genes Control Talc powder P-value TCA P-value Silver nitrate P-value
Gsr 20.00 ± 1.10 23.00 ± 2.62 <0.05 27.74 ± 3.16 <0.05 28.49 ± 4.10 >0.05
Gpx3 21.52 ± 1.04 22.86 ± 1.37 >0.05 28.05 ± 1.94 >0.05 29.66 ± 3.90 >0.05
Sod1 15.71 ± 0.85 18.27 ± 3.05 <0.05 19.87 ± 3.37 >0.05 20.58 ± 3.64 >0.05
Rai 25.97 ± 1.46 27.63 ± 2.52 >0.05 32.50 ± 1.12 >0.05 31.44 ± 2.02 >0.05
Hspa4 17.99 ± 1.22 18.46 ± 3.03 >0.05 21.34 ± 3.08 >0.05 21.49 ± 2.05 >0.05
Bax 21.85 ± 0.81 22.80 ± 2.15 >0.05 28.70 ± 2.15 >0.05 28.85 ± 3.25 >0.05
Bcl2 23.44 ± 0.73 24.80 ± 2.07 >0.05 30.07 ± 2.68 >0.05 29.72 ± 3.12 >0.05
Casp3 22.03 ± 1.08 23.92 ± 2.09 >0.05 28.83 ± 2.85 >0.05 29.20 ± 3.05 >0.05
Mdh1 18.08 ± 0.78 20.71 ± 2.59 >0.05 25.06 ± 3.21 >0.05 26.42 ± 4.14 >0.05
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*

bold values indicate the results statistically significant.

At the same time, expression levels of selected miRNAs were determined in the rat ovarian tissues. As shown in Table3, miRNA expression levels were increased in the talcum powder and TCA groups compared to controls but these results were not statistically significant. However, expression levels of miR-15b, miR-21 and miR-98 were raised in the group to which silver nitrate had been administered, and the results were statistically significant compared to the control group.

Table 3.

Expression levels of selected antioxidant, antiapoptotic and apoptotic miRNA genes

miRNAs Control Talc powder P-value TCA P-value Silver nitrate P-value
miR-98 24.33 ± 2.25 27.53 ± 5.08 >0.05 29.73 ± 5.21 >0.05 32.73 ± 1.15 <0.05
miR-15b 26.19 ± 3.44 29.76 ± 3.44 >0.05 30.22 ± 6.42 >0.05 34.23 ± 1.56 <0.05
miR-34a 26.15 ± 2.49 26.36 ± 3.74 >0.05 28.09 ± 4.59 >0.05 34.85 ± 3.32 >0.05
miR-21 16.22 ± 3.68 19.04 ± 5.04 >0.05 22.83 ± 5.96 >0.05 28.15 ± 3.57 <0.05
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*

bold values indicate the results statistically significant.

Discussion

Gene expression studies have been widely used in investigations which explore the molecular mechanisms of disease. Recent findings about the expression of mRNA and miRNA in ovarian tissue have provided much evidence which has enhanced our understanding of the molecular mechanism of the complex cellular events which occur in this tissue site (Chanyshev et al. 2014; Pereira et al. 2014). In parallel to these studies, miRNAs, which targets mRNA transcripts and thus regulates gene expression at post-transcriptional level, has attracted much attention in the last decade (Bartel 2004). miRNAs have known to play a key role in the carcinogenesis as well as various diseases (Lu et al. 2005; Calin & Croce 2006; Catto et al. 2011). Yet, limited number of studies have been focused on the molecular changes in rat ovary tissues induced by non-surgical sterilization methods. Particularly, molecular defence mechanisms induced by the chemicals used in non-surgical sterilization have not been well studied. Therefore, in the present study, we investigated the effects of talcum powder, TCA and silver nitrate in non-surgical sterilization by evaluating the alterations in the expression of apoptotic, anti-apoptotic and antioxidant mRNA and miRNA genes. The present study did not show a statistically significant increase in expression of either apototic or antioxidant associated genes.

Wehner (2002) postulated talcum powder as a non-carcinogenic agent used in sterilisation. Consistent with Wehner's results, no significant alteration was detected in expression of the selected apoptotic and antioxidant genes in rat ovarian tissue when talcum powder was used. In addition to the effect of the silver nitrate on proliferation has not been reported before in the literature. On the other hand, the carcinogenic activity of TCA has been reported (Pereira 1996). TCA is known to cause loss of heterozygosity on chromosome 6, demonstrated by Tao et al. (2000). Hypomethylation of proto-oncogenes such as c-myc and c-jun have been reported to be induced by TCA (Tao et al. 2000). In several cancer studies, the expression level of IGFII mRNA has been found to be increased and Tao et al. (2000) postulated in their study that expression of this gene was elevated in mouse liver tissue when promoted by TCA. The genomic region encoding IGFII mRNA transcript was found to be hypomethylated; thus, these events were suggested to be cause proliferation of cancer cells.

Furthermore, there is no existing study regarding the effects of TCA in rat ovary tissue. In our study, there were no significant alterations in the expression of apoptotic and antioxidant genes as a result of TCA administrations, suggesting that TCA may have tissue-specific effects and well tolerated in ovarian tissue. Acharya et al. (1997) showed that TCA and tert-Butyl alcohol (TBA) do not alter normal kidney histopathology at subtoxic doses. Therefore, our hypothesis seems to be logical in that TCA and TBA do not stimulate the activation of antioxidant and apoptotic genes. When we analyse the mRNA expression levels antioxidant genes, Gsr and Sod1 expressions these were found to be elevated in the group to which talcum powder was administered. In our previous study, similar to these results, expression of apoptotic and antiapoptotic genes was not altered. However, Sod1 activity was found to be increased (Kara et al. 2014). Also, in the group to which TCA was administered, only the levels of Gsr was found to be increased, and in the silver nitrate group, expression levels of antioxidant genes were similar to those in the control group. It is generally accepted that foreign agents cause stress in living organisms and this is associated with increased levels of free oxygen radicals. Cells will then respond to these events by invoking their antioxidant defence mechanisms. Sod1 and Gsr are a part of these defence mechanisms activated in response to free oxygen radicals (Rosen et al. 1993; Gao et al. 2012). According to our study results, we propose that antioxidant defence mechanisms are activated in response to the toxic effects of talcum powder and TCA. Silver, however, is an antioxidant itself and it may be that the non-toxic effects of the silver nitrate were observed because of this. Thus, silver nitrate administration did not change the expression of antioxidant genes in ovarian tissue. This result may give better understanding for the further studies.

Furthermore, expression levels of selected miRNAs were also evaluated and expression changes were only observed in silver nitrate group. While miR-21, miR-15 and miR-98 expression was increased in silver nitrate group, no expression changes were detected in terms miR-34a. This may imply that subtle selective miRNA changes are important as a response to injury rather than toxicity.

This is the first report that demonstrates the effects of talcum powder, TCA and silver nitrate on the expression of apoptotic, antiapoptotic, antioxidant genes and miRNAs in rat ovarian tissue. The results cannot be interpreted as a comment on carcinogenicity of these agents. Also, full toxicological studies are necessary to confirm safety, and actual free radical levels need to be explored. Protein expression levels analysed by Western blotting and quantified will give an idea as to how these findings are translated. Histopathology of ovarian tissue would also be of value, as would confirmatory analysis of cell lines. However, the findings suggest that different mechanisms - and risks - are involved in the use of these three sterilisation inducing agents.

Conflict of interest

There is no potential sources of conflict of interest to declare.

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