Pollution of the coastal environment and aquatic life with heavy metals and other chemical pollutants from uncontrolled anthropogenic activities has become a global public health issue. Heavy metal contamination of aquatic organisms would eventually translate to deleterious human health consequences through the food chain. Environmental and dietary exposure to HM have been associated with multiple organ and systemic toxicities including nephrotoxicity, hepatotoxicity, immunotoxicity, neurotoxicity, reproductive failure, genotoxicity and carcinogenesis (El-Sikaily A & M., 2021; Fuerst, 2019; Gade, Comfort, & Re, 2021; Kim, Ock, Moon, & Park, 2021; Lee, Min, & Min, 2020; Moody, Coca, & Sanders, 2018; Rehman et al., 2018). Mechanisms involving oxidative stress and oxidative DNA damage has been implicated as probable biological pathways of HM induced organ toxicities (Sharma, Singh, & Siddiqi, 2014). Exposure to HM contaminated mussels in relation to redox imbalance among women with recurrent pregnancy loss were assessed in this study.
Our study demonstrated that cadmium levels were higher than the EU maximum limits, lead levels below the maximum limit ("European Union (EU). Commission regulation (EC) No 1881/2006. Setting maximum levels for certain contaminants in foodstuff. L 364/ 5–24.," 2006) and elevated levels of metallothionein in mussel tissues studied. Our findings are in accordance with previous studies which had demonstrated higher levels of HM as cadmium, lead, copper, chromium, and zinc and measurable amount of metallothionein in mussel tissues collected from Abu Qir Bay (A. El Nemr, Khaled, Moneer, & El Sikaily, 2012; A. M. El Nemr, El Sikaily, & Khaled, 2007; Saad, El-Sikaily, & Kassem, 2017). Higher Cd content of mussels observed may result from pollution of coastal waters accruing from waste disposal from the community, diverse industrial process, sea transportation, and oil spills (Abdel Ghani, El Zokm, Shobier, Othman, & Shreadah, 2013). These activities have high potential to release waste containing heavy metals leading to HM contamination of coastal waters (Amalo LF et al., 2021). Heavy metals are not biologically biodegradable, hence form deposits and bioaccumulate in sediments, are absorbed and bioaccumulate in the bodies of marine life and through the food chain undergo environmental biomagnification and eventually reach humans (Rusydi et al., 2021). Mussels because of their slow mobility and filter feeding characteristics have the potential to accumulate heavy metals present in their environment. These characteristics may be responsible for high Cd levels observed in mussel tissues studied (B.Y. Kamaruzzaman et al., 2011). Mussels are commonly used to assess the eco-toxicological effects of the products released by anthropogenic activities. The concentration of metal in the tissue of mussels has been shown to increase concomitantly with the HM levels of the habitat (B.Y. Kamaruzzaman et al., 2011; Rusydi et al., 2021). Elevated MT in mussel may result from bioaccumulation of HM in the tissues. MT is a heavy-metal-binding protein mostly synthesized by aquatic organisms in response to the presence of heavy metals. (Chen et al., 2014). Due to their inducibility by heavy metals, metallothionein are usually considered as important specific biomarker to detect organism response to inorganic pollutants such as Cd, Hg, Cu, and Zn present in the aquatic environment (Amalo LF et al., 2021).
The biochemical and functional characteristics of metallothionein enables to protect cell structures from non-specific interactions with heavy metal cations and to detoxify metal excess penetrating into the cell (Chen et al., 2014; Rusydi et al., 2021).
Higher Cd, Pb and rbcMT were demonstrated in women with RPL compared to women without RPL. Elevated levels of Cd and Pb have been reported in women with recurrent miscarriages and abortions by previous studies (El-Maali NA, El-Baz MAH, Roshdy AS, & AF, 2015; MK., 2019; Omeljaniuk et al., 2018) and may highlight the possible role of subclinical Cd and Pb toxicity in the development of RPL. Studies showing evidence of detrimental effects of HM on fetal development and pregnancy outcome have been documented (K. Turan, A. Arslan, K. Uçkan, H. Demir, & C. Demir, 2019; Wang et al., 2020). HM as Cd, Hg, As and Pb has been shown to cross the placental barrier and accumulate in embryo tissues, inhibit intracellular calcium and magnesium ions Ca2 and Mg2, disrupt cell division, spindle formation and cell cycle which will eventually lead to cell death, fetal malformation and fetal loss (Wang et al., 2020). HM can also cause placental defects, leading to placental malfunction with a compromised capability to support embryonic growth. Cadmium and lead have been implicated in undermining ovum quality, normal ovum
Table 1
Comparison of HM, indices of oxidative stress, reproductive hormone and red cell indices in women without recurrent pregnancy loss (RPL) (control Group) and women with RPL (Groups I-III).
Index
|
Control
n = 18
|
Group I
n = 24
|
Group II
n = 18
|
Group III
n = 16
|
P-value
|
Control vs
Grp I
P-value
|
Control vs
Grp II
P-value
|
Control vs
Grp III
P-value
|
|
Heavy metals
|
|
Cd (µg/dl)
|
0.05 ± 0.05
|
0.80 ± 0.32
|
1.11 ± 0.12
|
1.65 ± 0.75
|
< 0.001*a
|
< 0.001*b
|
< 0.001*b
|
< 0.001*b
|
|
Pb (µg/dl)
|
3.72 ± 1.96
|
15.42 ± 3.52
|
18.55 ± 2.61
|
30.13 ± 6.54
|
< 0.001*a
|
< 0.001*b
|
< 0.001*b
|
< 0.001*b
|
|
Indices of oxidative stress
|
|
Catalase (IU/L)
|
449.70±
128.08
|
187.67±
92.66
|
126.35±
51.38
|
118.52±
43.96
|
< 0.001*a
|
< 0.001*b
|
< 0.001*b
|
< 0.001*b
|
|
GSH (mg/dl)
|
27.02 ± 3.85
|
25.60 ± 3.28
|
24.65 ± 3.35
|
20.78 ± 2.47
|
< 0.001*a
|
0.166
|
0.046*b
|
< 0.001*b
|
|
MDA (mg/dl)
|
3.04 ± 1.21
|
9.28 ± 3.06
|
9.22 ± 3.00
|
9.46 ± 2.90
|
< 0.001*a
|
< 0.001*b
|
< 0.001*b
|
< 0.001*b
|
|
rbcMT (mmol/gHb)
|
1.23 ± 0.32
|
3.28 ± 0.75
|
4.09 ± 1.05
|
4.65 ± 1.19
|
< 0.001*a
|
< 0.001*b
|
< 0.001*b
|
< 0.001*b
|
|
Reproductive hormone
|
|
|
|
|
P4 (ng/dl)
|
13.54 ± 11.91
|
2.18 ± 2.36
|
2.50 ± 2.24
|
1.83 ± 1.80
|
< 0.001*a
|
< 0.001*b
|
< 0.001*b
|
< 0.001*b
|
|
Red cell indices
|
|
|
|
|
Hb (g/dl)
|
11.59 ± 0.93
|
10.32 ± 0.82
|
9.87 ± 0.88
|
9.73 ± 1.27
|
< 0.001*a
|
< 0.001*b
|
< 0.001*b
|
< 0.001*b
|
|
MCV (µm3)
|
94.17 ± 14.57
|
76.24 ± 6.38
|
67.53 ± 4.17
|
65.41 ± 4.87
|
< 0.001*a
|
0.006*b
|
< 0.001*b
|
< 0.001*b
|
|
MCH (pg/cell)
|
29.08 ± 2.91
|
24.90 ± 2.41
|
22.82 ± 1.85
|
23.13 ± 2.57
|
< 0.001*a
|
< 0.001*b
|
< 0.001*b
|
< 0.001*b
|
|
Data presented as mean ± SD, * = indicates significant variations among the groups at P < 0.05, a = P values from Kruskal Wallis test, b= P values from Mann-Whitney U test, Cd = cadmium, Pb = lead, GSH = reduced glutathione, MDA = malondialdehyde, rbcMT = red cell metallothionein, P4 = progesterone, Hb = haemoglobin, MCV = mean corpuscular volume, MCH = mean corpuscular haemoglobin |
division, implantation of embryo leading to reduced pregnancy rates and an increase in fetal malformations, spontaneous abortion, and premature birth (Al-Saleh, Shinwari, Mashhour, Mohamed Gel, & Rabah, 2011). In addition, HM has been linked with disruption of the hypothalamic-pituitary-gonadal (HPG) axis, DNA methylation in neonates and chromosomal aberrations leadig to impaired embryonic development, and abortion (Li, Chen, Li, & Tollefsbol, 2019).
Positive associations were also observed between Cd and Pb, Cd with rbcMT, Pb with rbcMT and MDA with rbcMT respectively in women with recurrent pregnancy loss. Higher metallothionein observed in this present study corroborates our previous findings (Saad et al., 2017). Metallothionein has been shown to be a useful biomarker for the prediction of heavy metal toxicity and adverse biological outcome as its synthesis has been shown to be induced by exposure to HM. Upon heavy metals stimuli, metallothionein genes are rapidly transcriptionally activated and function in protecting cells from damage (Chen et al., 2014; Saad et al., 2017). Higher levels of Cd and Pb observed in women with RPL may have elicited higher MT levels also observed in these women. Induction of methalothionein synthesis by exposure to HM may also explain the significant positive associations observed between rbcMT and the HMs in this study. Strong correlation between MT expression and environmental heavy metal burden has been previously reported (A. M. El Nemr et al., 2007). Evidence from in vivo and invitro studies have shown that Metallothionein is thought to play a major role in systemic heavy metal detoxification and neutralization of ROS (Saad et al., 2017). MT function in heavy metal detoxification primarily depends on the high affinity binding between the heavy metals and MTs, leading to the sequestration of metals away from critical macromolecules (Chen et al., 2014; Saad et al., 2017). Besides HM, oxidative stress and lipid peroxidation has been shown to induce the expression of MT gene (Chen et al., 2014). This may therefore explain the positive association observed between MT and MDA in this study. Higher levels of MDA observed in women with RPL may be related to HM induced OS and subsequent peroxidation of membrane lipids and other biomolecule. Malondialdehyde (MDA) is formed from the breakdown of polyunsaturated fatty acids (PUFA) and it serves as a convenient index for determining the extent of lipid peroxidation (Nsonwu-Anyanwu, Ndudi Idenyi, et al., 2022; Saad et al., 2017). The observed positive association between Cd and Pb is an indication that these two metals function in synergy in the precipitation of multiple organ dysfunctions. The synergistic interactions between metals and minerals have been implicated in human diseases (Nsonwu-Anyanwu, Icha, et al., 2022).
Our study also demonstrated lower catalase, GSH and P4 in women with RPL compared to women without RPL. A similar observation has been made by previous studies (Al-Sheikh et al., 2019; Ghneim & Alshebly, 2016; Zejnullahu et al., 2021). Lower levels of GSH, catalase and P4 observed in women with RPL may be related to higher levels of Cd and Pb also observed in these women. This observation is supported by the concomitant negative associations observed between the HMs and these antioxidants (Cd and catalase, Cd and GSH and between Pb and GSH). Exposure to cadmium and lead has been shown to increase the generation of free radicals i.e., reactive oxygen species (ROS) such as hydrogen peroxide, hydroxyl and superoxide radicals and reactive nitrogen species (RNS). Enhanced generation of ROS can overwhelm cells intrinsic antioxidant defenses that may result in oxidative stress which has been reported to influence the female reproductive system adversely (Rehman et al., 2018). Reactive oxygen species affect multiple physiological processes from oocyte maturation to fertilization, embryo development and pregnancy outcome (Agarwal, Gupta, & Sharma, 2005; Khazaei & Aghaz, 2017). OS accruing from higher HMs results in increased lipid peroxidation, DNA damage leading to multiple organ dysfunctions (Amadi, Igweze, & Orisakwe, 2017). Depletion of systemic antioxidants and increased lipid peroxidation may be responsible for the elevated levels of MDA and concomitant decrease in GSH and catalase observed in women with RPL. Lower progesterone levels observed in this study is in agreement with observations from previous studies (Rivera-Nunez et al., 2021; Wang et al., 2020). Lower P4 levels has been attributed to dysregulated synthesis of oetrogen and progesterone induced by HM and OS (Ajayi, Charles-Davies, & Arinola, 2012; Kasim Turan, Ayse Arslan, Kazim Uçkan, Halit Demir, & Canan Demir, 2019). The decline in P4 could inhibit endometrial thickening and hinder uterine contractile function, likely leading to spontaneous abortion and adverse pregnancy outcomes (Rivera-Nunez et al., 2021).
Women with RPL also demonstrated lower Hb, MCV and MCH compared to their control counterparts. These red cell indices also associated negatively with Pb. A previous study has demonstrated lower MCH values in RPL compared to the controls (Al-Aghbary, 2017). Higher lead levels observed in women with RPL may explain lowered red cell indices observed. Chronic exposure to lead has been associated with reduction in red cell indices (Rahimpoor, Rostami, Assari, Mirzaei, & Zare, 2020). Lead is shown to induce changes in the composition of red blood cell (RBC) membrane proteins and lipids and to inhibit hemoglobin synthesis. The biochemical basis for this effect is not known but the effect may be accompanied by inhibition in the activity of sodium and potassium dependent ATPases (Glenn, Stewart, Schwartz, & Bressler, 2001). Contrary to our findings, other studies have reported comparable levels of Hb, MVC and MCH in women with or without RPL (Amadi et al., 2017; Wang et al., 2020).