KLK3 as a possible novel early biomarker of environmental exposure in young women living in Polluted area

Background: Kallikrein Related Peptidase 3 (KLK 3 ) is secreted by Skene's glands and, is considered ancestral homologues of the male prostate gland and has long been used as a biological marker of prostate cancer. Recent studies have shown that the synthesis of KLK 3 can be induced by steroid hormones in different tissues of women and in the menstrual cycle it seems to follow the cyclic variation of estradiol and progesterone. In addition, some environmental pollutants such as bisphenols, phthalate / DBP (DiButyl Phthalate) affect AR (Androgen Receptors) mediated signalling that directly regulates KLK 3 secretion. This suggests that environmental factors may play a role in KLK 3 secretion. Methods: 61 healthy women living in a high environmental impact (HEI) area, 58 healthy women living in a low environmental impact (LEI) area were evaluated on possible presence or changes of KLK 3 in serum at different phases of the menstrual cycle: blood samples taken in the follicular phase 5th-6th day, ovulatory phase 12th -13th day and luteal phase 19th -20th day of the menstrual cycle. For this aim, an ultra-sensitive kit for KLK 3 with a detection limit of 0.001 ng / mL was used. Results KLK 3 values showed two opposite peaks, women from HEI had a positive peak in the ovulatory phase with mean value of 9.90 ± 3.21 pg / mL while women from LEI had a negative peak in the ovulatory phase with mean values of 3.07 ± 1.49 pg / mL. Progesterone, showed a correlation with KLK 3 . Women from HEI had higher KLK 3 values on average and no signicant changes were evident between the three withdrawals in the different phases of the cycle. In contrast, women from LEI had a statistically signicant decrease between the follicular and ovulatory phase (p <0.0001) and a statistically signicant increase (p <0.0001) between the ovulatory and luteal phase. Conclusions: The data obtained seem to go beyond the known role of KLK 3 . The dosage of KLK


Background
Kallikreins are a subgroup of serine proteases responsible for various physiological functions and, as enzymes, are capable of cleaving peptide bonds in proteins. In humans, plasma kallikrein (KLKB 1 ) has no known homologs, whereas tissue peptidases-related kallikrein (KLKs) constitute a family of 15 highly conserved serine proteases. These genes are localized to chromosome 19q13, forming the largest contiguous group of proteases in the human genome. The 15 members of the kallikrein-related serine peptidase (KLK) have different tissue-speci c expression pro les. Initially, KLK 3 was believed to be absent from female tissues and uids. However, KLK 3 has been detected in some female tissues (including breast, ovarian and endometrial tissues) and body uids (serum, breast milk, amniotic uid). The presence of KLK 3 in these female tissues appears to be closely associated with the regulation of steroid hormones, particularly androgens and progestins. Estrogens themselves do not appear to affect KLK 3 regulation, but they may impair androgen-induced KLK 3 production. In tissues and uids, KLK 3 is found in two molecular forms: free KLK 3 is the enzymatically active form and in a complexed form bound to protease inhibitors. Recent studies [1] suggest that KLK 3 can no longer be considered only as a speci c prostate marker, but as a protein that could be produced by steroid hormone receptor cells under conditions of steroid hormone stimulation. Kallikreins are a rich source of disease biomarkers. The kallikrein locus is extraordinarily responsive to steroids and other hormones; in fact, at least 14 functional hormone response elements have been identi ed. A more complete understanding of the transcriptional regulation of kallikreins may help to formulate more concrete hypotheses about the physiological functions of kallikreins and their effectiveness as biomarkers. The best known of the kallikreins is KLK 3 , commonly known clinically as Prostate-Speci c Antigen, which is currently the serum biomarker for prostate cancer. The highest detectable proportion of the KLK 3 protein in serum is secreted by the prostate gland, and considerably higher concentrations, approximately 20,000 fold higher, can also be detected in human seminal plasma [2]. KLK 3 has been detected in salivary glands, brain, breast and other tissues, although at concentrations 100 times lower than the serum level [3]. Recent studies [4] report the presence of KLK 3 in female serum, in relation to PCOS [5] and hirsutism [6,7]. Of particular interest is vaginal cervical uid (CVF), which is a complex biological uid that hydrates the mucosa of the lower female reproductive system. Extensive proteomic and biochemical studies on CVF have revealed that it contains large amounts of endogenous proteases and protease inhibitors, including an abundance of several members of the tissue kallikrein-related peptidase (KLK) family. The roles of KLKs in the lower female reproductive system are not fully understood, their activation is pH-dependent and there are also various other modes of regulation in the vagina. Roles have been postulated in physiological related to antimicrobial processes, vaginal and cervical epithelial desquamation, sperm transport and foetal membranes processing of, as observed in premature membranes rupture, through binding of known and unknown kallikrein substrates [8]. Despite their presence in human tissues and uids, expression levels of KLKs vary widely, with highest expression observed in reproductive-related tissues and uids. Zarghami and colleagues [9] report that KLK 3 is secreted cyclically during the menstrual cycle and appears to follow the peak concentration of Progesterone, with a delay of 10-12 days. Serum KLK 3 concentrations are highest during the mid-to-late follicular phase, fall continuously with a half-life of 3-5 days between the late follicular phase and mid-cycle and reach a minimum during the mid luteal phase. In many tissues, kallikrein expression is regulated by steroid hormones. In fact, the kallikrein locus is exceptionally responsive to hormones because each kallikrein is up-regulated by multiple hormones. For this reason, many researchers use kallikreins as a marker of hormone receptor activity [10,11]. Progesterone regulates kallikrein expression in various tissues, sometimes through direct binding of the PR (Progesterone Receptor) to kallikrein promoters. KLK 2 and KLK 3 are both up-regulated by Progesterone in breast cancer cell lines [12]. KLK 3 is directly related to androgen and estrogen receptor up-regulation.
Several data suggest that various kallikreins are regulated by steroid hormones, in particular androgens stimulate the KLK 3 -secreting cell line while estrogens stimulate the KLK 10, KLK 11 and KLK 14 -secreting cell line. Furthermore, KLK 3 expression is down-regulated by combined stimulation, con rming that estrogens can antagonise and block androgen receptor activity [13]. Therefore, acquiring more detailed information about the mechanisms regulating kallikrein expression should be a priority and the kallikrein locus could be an important model in the era of genome-wide analyses. Lorenzetti and colleagues [14] highlight the ability of some environmental pollutants to stimulate prostatic epithelial cells in vitro, resulting in KLK 3 secretion. In addition, other chemicals such as bisphenols, phthalate / DBP (DiButyl Phthalate) affect AR (Androgen Receptors) mediated signalling that directly regulates KLK 3 secretion. This suggests that environmental factors may play a role in KLK 3 secretion [15]. In order to suggest a new bio-marker related to reproductive health effects caused by environmental factors, we will try to assess how the serum concentration of KLK 3   Participants in the study were selected by examining the quality and quantity of environmental pollution in the area in which they lived, and they were all of childbearing age. A speci c "participant form " was proposed, in which anamnestic and clinical data are reported in order to create a database. Participants reported symptoms and signs of any medical conditions, hirsutism, acne and anamnestic data related to their lifestyle, use and/or abuse of alcohol, smoking and possible drug use. Participants had no major chronic diseases and had resided permanently in the selected areas for at least 5 years, were not professionally exposed to risk factors and had not taken oral contraceptive pills for at least 2 years. They denied having used drugs in the 12 months before the blood draw. Participants were asked about their age at menarche, whether they were nulliparous/multiparous, and whether they had experienced spontaneous/voluntary abortions. The participants had normal menstruation, the duration of the whole cycle varied from 28 to 30 days; and regularity, volume and duration were regular. In addition, BMI (body mass index), waist circumference, waist-to-hip ratio and Ferriman-Gallwey score for hirsutism were calculated.
Blood samples were collected during the 3 phases of the menstrual cycle, namely on days 5-6 (follicular phase), 12-13 (ovulatory phase) and 19-20 (lutein phase). KLK 3 was assayed in all three sera sample and progesterone was assayed on the third sample.
The method we have chosen for the KLK 3 assay in serum is the Access Hybritech KLK 3 assay, developed by Beckman Coulter, which is a two-site immunoenzymatic assay, also known as sandwich immunoassay, using the principle of chemiluminescence and producing light directly proportional to the concentration of KLK 3 in the sample. We used the UniCel DxI 600 Access Immunoassay System. The whole database, covering all participants, was subjected to a statistical analysis for each area and for the entire enrolled population. Assuming a signi cant difference between the groups belonging to the two selected areas, differences were found between the 61 participants from the high environmental impact area and the 58 participants from the low environmental impact area. A statistical analysis for each area and for the whole enrolled population was performed across the two groups using the Student's "t"-Test.
Signed informed consent was received from all participants in this study, in accordance with the ethical principles of experimentation (institutional or regional) and the Declaration of Helsinki of 1975, revised in 2000. Table 2 shows the variables of the two groups under examination; data were obtained from the forms completed by the participants and from the clinical evaluation performed by our collaborators.  3 and Progesterone values performed in the luteal phase, showed a signi cant correlation (p < 0.001). In fact, the mean value of progesterone in the high environmental impact group was 6.67 ± 5.73 ng/mL, while in the low environmental impact group it was 14.1 ± 6.19 ng/mL. We noted that the progesterone limit value of 14.1 ng/mL corresponded to an opposite trend in KLK 3 concentrations. For this value, 90.3% of the participants belonging to the high environmental impact group had a positive KLK 3 peak in the ovulatory phase and 93.6% of the participants belonging to the low environmental impact group had a negative peak in the ovulatory phase (Fig. 2).  [17][18][19]. Filella and colleagues [19] in 1996 detected the presence of KLKin 58% of female sera, Escobar-Morreale and colleagues [20] in 1998, detected the presence of 36.4 % KLK 3 in the serum of women with normal menstrual cycles and Mannello and colleagues [21] in 2001, did not detect the presence of KLK 3 in female serum. Normal serum levels of KLK 3 in women range from 0.02-0.06 ng/mL [22]. In this group, the highest concentrations of KLK 3 were found during days 5-6 of the menstrual cycle (7.18 ± 1.57 pg/mL), a second, smaller peak was observed on days 19-20 (6,29 ± 1,97 pg / mL). Yu and colleagues found low levels of KLK 3 during days 10-23 and a peak at the end of the cycle or at the beginning of the next cycle [23]. For this group, the peak was also seen at the beginning and at the end of the cycle in our study.

Results
Changes in KLK 3 values were statistically signi cant: comparison of values between the follicular phase (7.18 ± 1.57 pg/mL) and the ovulatory phase (3.07 ± 1.49 pg/mL) showed a signi cant decrease with p < 0.0001; comparison of values between the ovulatory phase (3.07 ± 1.49 pg/mL) and the lutein phase (6.29 ± 1.97 pg/mL) showed a signi cant increase with p < 0.0001 (Fig. 4).

Female participants living in high environmental impact area
Female study participants were aged from 22 to 37 (28.66 ± 4.43). Out of a total of 61 female participants included in the study, 83.6 % had detectable KLK 3 . In this group, the highest concentrations of KLK 3 were found on days 12-13 of the menstrual cycle (9.90 ± 3.21 pg/mL). Changes in KLK 3 values in the three cycle phases: follicular phase 8.95 ± 2.90, ovulatory phase 9.90 ± 3.21 and lutein phase 9.63 ± 2.87, were not statistically signi cant (Fig. 5).
In this group, progesterone values were detected on days 19-20 of the cycle with values ranging from 0.4 to 21.2 ng/mL (6.67 ± 5.72 ng/ml). We found statistically signi cant (p < 0.0001) changes in progesterone values among the two groups studied, depending on their residence (Fig. 6).

Figure 6: mean and standard deviations of progesterone values in the two groups
Progesterone is the main endocrine product of the corpus luteum during the menstrual cycle [9]. In vitro studies have shown that sera obtained during the menstrual cycle can stimulate KLK 3 production in a breast cancer cell line (T-Line 47D). Measurement of KLK 3 protein and KLK 3 mRNA levels showed that the ability of serum to induce KLK 3 production in cancer cells was proportional to serum progesterone levels, with the greatest stimulation occurring with serum containing more progesterone (days 22-24 of the menstrual cycle). With luteal phase serum, KLK 3 mRNA expression was signi cantly increased [9].
In 79.8% of all participants, KLK 3 is detectable in serum and is expressed in the three phases of the menstrual cycle: in the follicular phase the minimum value is 3.52 pg / mL and the maximum 17.74 pg / mL, in the ovulatory phase the minimum value is 0.11 pg / mL and the maximum value is 20.36 pg / mL and in the luteal phase the minimum value is 1.12 pg / mL and the maximum value is 19.24 pg / mL. The concentration of KLK 3 in the blood serum of the young nulliparous women who participated in this work showed a negative peak in the ovulatory phase in 46.3% and a positive peak in the ovulatory phase in 53.7%. On the other hand, in the multiparous it showed a negative peak in ovulatory phase in 86.3%. As previously reported, comparison of KLK 3 values with the anamnestic data showed a signi cant change (p < 0.05) between the two groups of participants for alcohol use and previous abortions, semeiotic evaluations between the two groups showed a signi cant change (p < 0.05) for the waist / hip ratio. Changes in KLK 3 concentrations have been reported in several studies re ecting serum progesterone levels, albeit with a delay of 12 to 16 days. In a biphasic pro le, KLK 3 is produced in target tissues in response to increased progesterone levels in the ovulatory phase, KLK 3 diffuses into the blood and is detected in the luteal phase. Progesterone spikes during the luteal phase are able to provide a spike in KLK 3 in the rst follicular phase of the next cycle [4,9]. The interpretation of the negative and positive peaks of KLK 3 in serum found in the serum of participants from LEI area and HEI area respectively is different, perhaps some mechanism of KLK 3 secretion is independent of progesterone. In the HEI area participants were found to be higher on average with insigni cant variation between the three phases of the cycle, it is conceivable that other stimuli, such as environmental factors, prompt a continuous secretion by the epithelial cells of the glands by Skene. Indeed, in other studies it has been shown that prostate epithelial cells respond to the stimulation of certain pollutants [14]. The concentrations of KLK 3 in the blood serum of participants living in LEI area showed a negative peak in the ovulatory phase with signi cant changes (p < 0.0001) between the follicular and ovulatory phase and the ovulatory and luteal phase, this cyclicality seems to respond to endogenous stimuli.

Conclusions
This study highlights an "interference" in the secretion of KLK 3 , in women who permanently reside in HEI area. Environmental pollutants in the HEI area may exert an endocrine interference that stimulates the Skene's glands, in fact during the menstrual cycle the concentrations of KLK 3 are higher and with minimal uctuations. A greater understanding of the functional roles of KLK 3 in the female reproductive system could lead to new diagnostic and therapeutic modalities for conditions such as vaginal infections, vaginal atrophy, and a probable role on sperm and endometrium.
This pilot study suggest a new role of KLK 3 in women. Its changes during the phases of the menstrual cycle demonstrate an action different other than simple secretion. The data obtained seem to go beyond the functions advanced by other authors on its role in antimicrobial processes, vaginal and cervical epithelial desquamation, and spermatozoa transport. The correlation with progesterone in the different phases of the menstrual cycle of young women and the different peaks in the ovulatory phase in the two study groups for areas with different environmental impact, allows us to consider the cyclic secretion and the concentration of KLK 3 as a new marker of environmental exposure in women. However, further studies, larger numbers and more homogeneous age group sampling will be needed to better understand the functions of KLK 3 in a woman's serum on the different days of the cycle.

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