We hypothesized that the environmental contaminant benzene and the plant
antioxidant quercetin may affect ovarian cell functions and that quercetin
could offer protection against the adverse effects of benzene. This study
aimed to examine the action of benzene, quercetin, and their combination on
porcine ovarian granulosa cell functions. We elucidated the effects of
benzene (20
Benzene is a common industrial chemical, a component of gasoline, and a constituent of engine emissions and tobacco smoke (Kalf, 1987). It is a known human carcinogen and numerous case reports and epidemiological studies have provided evidence of a causal relationship between exposure of females to benzene and abnormal menstrual cycles, severe bleeding, convulsions, and a higher rate of aborted pregnancies. The influence of benzene on the female reproductive system, in particular on fertility (Mukhametova and Vozovaya, 1972; Vara and Kinnunen, 1946) and the menstrual cycle (Michon, 1965), has been previously examined, but these studies did not generate definitive conclusions. The mechanisms underlying the action of benzene on the female reproductive system remain unclear. Oil-related contaminants can affect the female reproductive system by direct action on ovarian cells via ovarian functions regulated by upstream hypothalamo-hypophysial system (Sirotkin and Harrath, 2014). The target of benzene action remains to be established. If the direct action of benzene on the ovary could be detected, its mechanisms would require further examination because ovarian functions are regulated by ovarian cell proliferation, apoptosis, and secretory activity (Sirotkin, 2014). To our knowledge, there have been no studies exploring the mechanisms underlying the effects of benzene on the female reproductive system. Therefore, there is an urgent need to establish strategies for neutralizing the negative effects of benzene on the female reproductive system. Some plant molecules can protect cells from the negative effects of environmental stressors. For example, the flavonoid quercetin, which is present in fruits, vegetables, and beverages, can improve animal reproductive function (Dhawan et al., 2002) via anticancer (Aalinkeel et al., 2008; Jeong, 2008; Jeong et al., 2009; Warren, 2009), antihypertensive (Mackraj et al., 2008), anti-inflammatory (Kumazawa et al., 2006), and antimicrobial (Davis et al., 2008) effects. It may do so, for example, by suppressing tumour cell proliferation (Jeong, 2008; Warren, 2009). The action of quercetin on healthy ovarian cells has been demonstrated previously on rabbit (Leśniak-Walentyn et al., 2013), mouse (Shu et al., 2011) and bovine (Tarko et al., 2018) ovaries; however, the action of quercetin on porcine reproductive processes and the ability of quercetin to protect these processes against environmental contaminants, including benzene, have not yet been studied.
The main aim of this study was to evaluate the effect of benzene, quercetin, and their combination on basic ovarian cell functions (proliferation, apoptosis, hormone release) and to examine the possible protective effect of quercetin against benzene action.
We obtained 12 ovaries from 3- to 5-month-old Landrace breed specimens at the follicular stage of the estrous cycle from the slaughterhouses of the National Agricultural Food Centre in Nitra and in Stará Myjava.
The ovarian granulosa cells were prepared according to Tarko et al. (2017).
After 96 h of culture, when the cells created a monolayer of 75 %, the
medium was replaced with a fresh one that was supplemented with one of the
following treatment conditions: (1) control groups without treatment of
quercetin (Q) (for research use only, sc-206089A, Santa Cruz
Biotechnology) extract (AppliChem GmbH, Germany; 0
Proliferation (PCNA) and apoptosis (BAX) markers were detected via
immunocytochemistry using the method described by Osborn and Isenberg (1994)
and Tarko et al. (2017). The presence of markers of proliferation and
apoptosis was detected by immunocytochemistry (Osborn and Isenberg,
1994). After washing and fixation, the cells were incubated in blocking
solution (1 % goat serum (Santa Cruz Biotechnology Inc., Dallas, TX, USA)
in PBS) at room temperature for 1 h to block non-specific binding of the
antiserum. The cells were incubated at the presence of monoclonal antibodies
against either PCNA (dilution
Concentrations of progesterone, oxytocin, and prostaglandin F were determined
in 25–100
The EIA for P, OT, and PGF was based on the paper of and performed in accordance
to Prakash et al. (1987) with our slight modifications (Kotwica et al., 1993, 1994). The cross-reactivity of the antisera used
against OT and P4 were previously reported by Kotwica et al. (1993, 1994), and those against plasma 13,14-dihydro-15-keto alpha (PGFM) were reported by Homanics and
Silvia (1988). The range of the standard curve, the intra- and inter-assay
coefficients of variation, and the relationship between the added and
measured hormone concentrations (
We performed three independent repetitions for ICC experiments and four EIA
repetitions. In the independent experiments, each experimental group was
represented by four culture wells for enzyme immunoassays or one
chamber slide well for immunocytochemistry according Tarko et al. (2017)
using a two-way ANOVA followed by Dunnett's test. In this paper, only the
comparison between the following selected groups has been presented: (1) the
differences between cells without and with quercetin extract addition at
three different levels without benzene, (2) the differences between cells
with or without benzene, (3) the differences between cells without and with three different concentrations of quercetin extract with the simultaneous addition of
benzene. Values are presented as mean
The administration of 20
Effects of benzene, quercetin extract, and their combination on
cell proliferation in cultured porcine ovarian granulosa cells. Parts marked “a” show the effect
of quercetin extract (significant difference (
The administration of 20
Effects of benzene, quercetin extract, and their combination on
apoptosis in cultured porcine ovarian granulosa cells. Parts marked “a” show the effect of
quercetin extract (significant difference (
B (20
Effects of benzene, quercetin extract, and their combination on
the release of progesterone in cultured porcine ovarian granulosa cells. Parts marked “a” show the effect of quercetin extract (significant difference (
B (20
Effects of benzene, quercetin extract, and their combination on
the release of oxytocin in cultured porcine ovarian granulosa cells. Parts marked “a” show the
effect of quercetin extract (significant difference (
B (20
Effects of benzene, quercetin extract, and their combination on
the release of prostaglandin F in cultured porcine ovarian granulosa cells.
Parts marked “a” show the effect of quercetin extract (significant difference (
Our results showed that the administration of benzene stimulated proliferation and apoptosis in porcine ovarian granulosa cells. This is the first evidence for direct action of benzene on the ovary. As PCNA is a marker of the S phase of mitosis (Connolly and Bogdanffy, 1993), we expect that benzene may target this phase of the cell cycle. Moreover, the stimulatory action of benzene on cell proliferation observed in our experiments could explain the ability of benzene to induce malignant transformations characterized by increased cell division (Arp et al., 1983). BAX is a known regulator and marker of cytoplasmic apoptosis (Gaumer et al., 2000); therefore, it might be hypothesized that benzene promotes this kind of apoptosis. Furthermore, the ability of benzene to promote both proliferation and apoptosis suggests that benzene can increase ovarian cell turnover. The physiological significance of this effect, as well as the interrelationships between proliferation and apoptosis in ovarian cells when influenced by benzene, requires further clarification. We observed that benzene exerted an inhibitory effect on progesterone and prostaglandin F release and a stimulatory effect on oxytocin release. This is the first demonstration of the effect of benzene on the release of ovarian hormones. These hormones are known regulators of ovarian cell proliferation, apoptosis, steroidogenesis, folliculogenesis, and fecundity (Sirotkin, 2014). Thus, it might be proposed that the previously reported adverse effects of benzene on the female reproductive system (Vara and Kinnunen, 1946; Michon, 1965; Mukhametova and Vozovaya, 1972) may be due to the direct action of benzene on the release of ovarian hormones, which in turn regulate ovarian cell proliferation, apoptosis, and other reproductive processes. Altogether, our study is the first to demonstrate the potential ability for benzene to directly affect ovarian cells by promoting proliferation, apoptosis, and oxytocin release and by inhibiting the release of progesterone and prostaglandin F.
Our observations demonstrated that quercetin inhibits proliferation and apoptosis and also stimulates oxytocin (but not progesterone and prostaglandin) release in porcine ovarian cells.
The studies of Gao et al. (2012) and Ren et al. (2015) observed that quercetin exerted an inhibitory effect on proliferation and a supportive effect on apoptosis in human ovarian cancer cells. Similarly, another study on patients with pancreatic cancer (Angst et al., 2013) suggests a possible benefit of quercetin. However, these results contradict the findings of Leśniak-Walentyn et al. (2013), who demonstrated increased proliferation (Shu et al., 2011) and decreased apoptosis in rabbit (Shu et al., 2011; Tarko et al., 2018) and decreased apoptosis in bovine (Tarko et al., 2018) ovaries cultured with quercetin. The study (Tarko et al., 2018) also demonstrated an inhibitory effect of quercetin on progesterone release. Our results are in partial agreement with all of these studies. The differences in the characteristics of quercetin action on ovarian cell proliferation and apoptosis could be explained by the differences in species (human, rabbits, bovine, porcine), cell health (normal and malignant cells), and markers (PCNA, caspase-3, terminal deoxynucleotidyl transferase, BAX) measured or used in different studies. Oxytocin promotes cell proliferation and steroid hormone release by ovarian cells (Berisha and Schams, 2005; Niswender et al., 2007; Skarzynski et al., 2008; Sirotkin, 2014) and apoptosis in neonatal ovaries (Marzona et al., 2001). During luteolysis, oxytocin as a luteotropic hormone can oppose the luteolytic action of prostaglandin F2 alpha (Gimpl and Fahrenholz, 2001). Our study did not show an association between quercetin and the proliferation and apoptosis or the release of any hormone, which suggests that quercetin probably does not affect proliferation and apoptosis via the release of progesterone, oxytocin, PGF, or PGM. This is the first demonstration of the effect of quercetin on healthy bovine ovaries. Nevertheless, the action of quercetin on ovarian cell proliferation, apoptosis, and oxytocin release that was observed here suggests the involvement and applicability of quercetin in the regulation of porcine and human reproductive processes, including fecundity. It suggests, that the addition of quercetin in animal feed could help improve porcine fertility. However, this hypothesis requires validation with adequate in vivo studies.
Some negative effects of environmental stressors on ovarian functions could be prevented or neutralized by certain plants that contain antioxidants and adaptogens (Ungvary et al., 1981; Liang and Yin, 2010). Animal experiments suggest that quercetin, due to its anti-oxidative and anti-apoptotic characters, may provide effective protection against the toxic effects of cadmium (Bu et al., 2011) and dimethyl sulfoxide (Cao et al., 2007). We failed to find any previous research on the potential protective effect of quercetin against the action of benzene or other petrochemical environmental contaminants.
In our experiments, quercetin supported the effect of benzene on progesterone release and did not modify the effect of benzene on proliferation, apoptosis, and oxytocin and prostaglandin F release. Therefore, our observations provide the first evidence that quercetin does not appear to protect ovarian cells against the negative effects of benzene on ovarian cell proliferation (accumulation of PCNA), apoptosis (BAX), and the release of these hormones. Therefore, quercetin cannot be considered protective against benzene action with respect to these processes. Consequently, we may not expect quercetin to protect against benzene, which may induce carcinogenesis by promoting cell division (Arp et al., 1983). Moreover, the cumulative action of quercetin and benzene on ovarian cell progesterone output suggest that quercetin does not protect but rather may even promote the negative effect of this contaminant.
In summary, our observations demonstrate the direct effects of both benzene and quercetin on basic porcine ovarian cell functions (proliferation, apoptosis, and secretory activity). Benzene promoted ovarian cell proliferation, apoptosis, and oxytocin release, and it inhibited progesterone and prostaglandin F release. Quercetin inhibited proliferation and apoptosis, but it stimulated oxytocin. Therefore, quercetin may prove potentially useful in the control of animal and human reproduction, including to enhance fertility or treat reproductive disorders. This is the first demonstration of how quercetin can modify the effect of benzene on ovarian cell function. However, in this paper, quercetin did not prevent but rather promoted benzene action on ovarian function. Therefore, it cannot be used for the prevention of the effects of benzene on these processes. The hypotheses arising from this study require further verification with appropriate in vivo studies.
The data used in this paper are available on request from the corresponding author (tarko.adam.000@gmail.com).
AT, AS, KJ, SH, AV, AHH, JK, and AVS assisted in conducting in vitro experiments in the laboratory. AS, KJ, SH, AV, AA, JK, and AB provided technical support. AT, SA, AA, and AVS prepared the paper, and KJ and AVS managed the corrections to the paper.
The authors declare that they have no conflict of interest.
The authors would like to thank to Zofia Kuklova and Katarina Tothova (National Agricultural and Food Centre in Lužianky) for technical assistance. This research was supported by the grants APVV–0854–11, APVV–0404–11, APVV–15–0296, and UGA VIII/26/2017. The authors would like to express their appreciation to the Deanship of Scientific Research at King Saud University for funding this research group RG-254.
This research has been supported by the Agentúra Ministerstva Školstva, Vedy, Výskumu a Športu SR (grant no. 0854–11); the Agentúra Ministerstva Školstva, Vedy, Výskumu a Športu SR (grant no. 0404–11); the Agentúra Ministerstva Školstva, Vedy, Výskumu a Športu SR (grant no. 15–0296), the Univerzitná grantová Agentúra (grant no. VIII/26/2017); and the Deanship of Scientific Research at King Saud University, which funded this research group RG-254.
This paper was edited by Manfred Mielenz and reviewed by two anonymous referees.