Kaı̈s H Al-Gubory

Environmental pollutants and lifestyle factors induce oxidative stress and poor prenatal development

Developmental toxicity caused by exposure to a mixture of environmental pollutants has become a major health concern. Human-made chemicals, including xenoestrogens, pesticides and heavy metals, as well as unhealthy lifestyle behaviours, mainly tobacco smoking, alcohol consumption and medical drug abuse, are major factors that adversely influence prenatal development and increase susceptibility of offspring to diseases. There is evidence to suggest that the developmental toxicological mechanisms of chemicals and lifestyle factors involve the generation of reactive oxygen species (ROS) and cellular oxidative damage. Overproduction of ROS induces oxidative stress, a state where increased ROS generation overwhelms antioxidant protection and subsequently leads to oxidative damage of cellular macromolecules. Data on the involvement of oxidative stress in the mechanism of developmental toxicity following exposure to environmental pollutants are reviewed in an attempt to provide an updated basis for future studies on the toxic effect of such pollutants, particularly the notion of increased risk for developmental toxicity due to combined and cumulative exposure to various environmental pollutants. The aims of such studies are to better understand the mechanisms by which environmental pollutants adversely affect conceptus development and to elucidate the impact of cumulative exposures to multiple pollutants on post-natal development and health outcomes. Developmental toxicity caused by exposure to mixture of environmental pollutants has become a major health concern. Human-made chemicals, including xenoestrogens, pesticides and heavy metals, as well as unhealthy lifestyle behaviors, mainly tobacco smoking, alcohol consumption and medical drug abuse, are major factors that adversely influence prenatal development and increase the susceptibility of offspring to development complications and diseases. There is evidence to suggest that the developmental toxicological mechanisms of human-made chemicals and unhealthy lifestyle factors involve the generation of reactive oxygen species (ROS) and cellular oxidative damage. Overproduction of ROS induces oxidative stress, a state where increased generation of ROS overwhelms antioxidant protection and subsequently leads to oxidative damage of cellular macromolecules. Exposure to various environmental pollutants induces synergic and cumulative dose-additive adverse effects on prenatal development, pregnancy outcomes and neonate health. Data from the literature on the involvement of oxidative stress in the mechanism of developmental toxicity following in vivo exposure to environmental pollutants will be reviewed in an attempt to provide an updated basis for future studies on the toxic effect of such pollutants, particularly the notion of increased risk for developmental toxicity due to combined and cumulative exposure to various environmental pollutants. The aims of such studies are to better understand the mechanisms by which environmental pollutants adversely affect conceptus development and to elucidate the impact of cumulative exposures to multiple pollutants on postnatal development and health outcomes.

Developmental changes in antioxidant enzymatic defences against oxidative stress in sheep placentomes.

Early pregnancy is susceptible to oxidative stress, and thus characterisation of antioxidant systems and pro- and anti-apoptotic pathways would improve understanding of placental development and function. We aimed, therefore, to determine the activities of the antioxidant enzymes, copper/zinc-superoxide dismutase (SOD1), manganese-SOD (SOD2), catalase (CAT), glutathione (GSH) peroxidase (GPX) and GSH reductase (GSR); and to quantify the expression of BAX and MCL1 proteins in relation to the developmental changes in antioxidant defences in sheep placentomes sampled on days 35, 55 and 80 of pregnancy. Placentome progesterone content was analyzed to determine steroidogenic capacity. Malondialdehyde (MDA) and protein carbonyl were quantified in placentomes as biomarkers of lipid peroxidation and protein damage respectively. Placentome tissues demonstrated significantly increased content of progesterone and MDA at day 80 of pregnancy and protein carbonyl as early as day 50 of pregnancy. Progesterone and MDA contents were not different between days 35 and 55 of pregnancy. While SOD1 and CAT activities did not alter significantly, SOD2 activity decreased from days 35 to 55. GPX activity increased from days 35 to 55 and increased further to day 80 of pregnancy. GSR activity increased from days 35 to 55 of pregnancy. BAX protein expression decreased, while MCL1 increased from days 35 to 55 and 80 of pregnancy. The increased GPX activity was associated with a decrease in the BAX/MCL1 protein expression ratio. Changes in the antioxidant enzymatic defences could be a part of placentome adaptation to reactive oxygen species-induced oxidative stress at specific early developmental stages of pregnancy.

Regulation of Key Antioxidant Enzymatic Systems in the Sheep Endometrium by Ovarian Steroids

Reactive oxygen species (ROS) and their control by antioxidant enzymes are involved in the physiology of the female reproductive system. Thus, it is important to understand the regulation of key antioxidant enzymatic pathways. The roles of estrogen and progesterone in regulating the physiological functions of the endometrium have become central dogma. We examined the effects of ovarian steroids on superoxide dismutases (SOD1 and SOD2), catalase (CAT), glutathione peroxidase (GPX), and glutathione reductase (GSR) activities in the aglandular caruncular and glandular inter-caruncular endometrial tissues of ovariectomized (OVX) ewes and in OVX ewes treated with estradiol (E2), progesterone (P4), or both hormones according to schedules designed to produce physiological changes of these hormones during the estrous cycle. The activities SOD2, CAT, GPX and GSR in both endometrial tissues were unaffected by P4 treatment. The activity of SOD1 in the aglandular tissue was unaffected by P4 treatment, however this treatment decreased SOD1 activity in the glandular tissue (P < 0.01). Treatment with E2, either alone or in combination with P4, decreased SOD1 (P < 0.01), CAT (P < 0.01) and GPX (P < 0.05) activities in both endometrial tissues. The activity of GSR decreased only in the glandular tissue (P < 0.05) after E2 treatment, either alone or in combination with P4. No change in SOD2 activity was detected in both endometrial tissues after administration of E2, P4 or both hormones. This study provides the first firm evidence for the role of ovarian steroid hormones in the regulation of the activities of key antioxidant enzyme in the endometrium of female mammals.

Ovine corpus luteum proteins, with functions including oxidative stress and lipid metabolism, show complex alterations during implantation

Progesterone (P(4)) secreted by the corpus luteum (CL) is critical for in utero embryo survival and development, although CL proteins are key regulatory factors during the luteal phase. We, therefore, characterised protein expression patterns in ovine CL of pregnancy (days 12, 16 and 20) compared with those of controls, CL of oestrous cycle (days 12 and 16), using two-dimensional gel electrophoresis (2DE) gel-based proteomics. Proteins in 24 significantly altered spots were identified by tandem mass spectroscopy. At the time of embryo implantation (day 16), 77 spots were up-regulated and 101 spots were down-regulated in CL of pregnancy compared with regressed CL. Vimentin, lamin A/C (LMNA), [Mn] superoxide dismutase (SOD2), isocitrate dehydrogenase 1, annexin A1 and elongation factor Tu, mitochondrial (TUFM) altered during CL regression, whereas glutathione S-transferase A1, apolipoprotein A-1, myxovirus resistance protein 1, ornithine aminotransferase and enoyl-CoA hydratase, mitochondrial (ECHS1) tended to be altered during CL maintenance. biliverdin reductase B (BLVRB), FDXR, guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-2 (GNB2) and cytochrome b-c1 complex subunit 1, mitochondrial (UQCRC1) showed divergent expression during CL regression and maintenance. The expression of two representative proteins, SOD2 and BLVRB, by western blot increased in CL of non-pregnant ewes on day 16 compared with that on day 12. SOD2 and BLVRB were localised in the large and small luteal cells and endothelial cells of CL over peri-implantation periods. 2DE gel and mass spectrometry have been used, for the first time, to study ovine CL function. We have identified proteins involved in key pathways, including oxidative stress, steroidogenesis, signal transduction and apoptosis, which have not previously been associated with changes occurring in the CL during the peri-implantation period. These proteins are most likely involved with mechanisms allowing the CL to produce P(4) during early pregnancy.

Corpus luteum derived copper, zinc-superoxide dismutase serves as a luteinizing hormone-release inhibiting factor in sheep.

In the present study, we report the purification and characterization of a polypeptide from the sheep corpus luteum of pregnancy with a potent luteinizing hormone-release inhibiting factor (LH-RIF) bioactivity that stained as a single band in SDS-PAGE with an apparent molecular mass of 16000 Da. The amino acid sequences obtained after sequence analysis of peptides derived from the trypsin digestion of LH-RIF were subjected to a protein data bank search and were found to be identical with regions of sheep copper, zinc-superoxide dismutase (Cu,Zn-SOD). The measured mass of LH-RIF (15604.2+/-1.9 Da) was found to be similar to the theoretical mass of sheep Cu,Zn-SOD (15603.5 Da), with a disulfide bond and N acetylated alanine at the N-terminus. The inhibitory action of Cu,Zn-SOD on pulsatile LH secretion would suggest that this antioxidant may play an important role, either independently or in concert with some neurotransmitters, in the neuroendocrine regulation of sheep female reproductive function.

Intracerebroventricular administration of copper-zinc superoxide dismutase inhibits pulsatile luteinizing hormone secretion in ovariectomized ewes

Intracerebroventricular (i.c.v.) injection of an inhibitor of nitric oxide synthase (NOS) abolishes pulsatile luteinizing hormone (LH) secretion. It has been demonstrated that structural and functional analogs of copper-zinc superoxide dismutase (Cu,Zn-SOD) inhibit neuronal NOS. The present study examined the ability of Cu,Zn-SOD to affect pulsatile LH release in the ewe. Bovine Cu,Zn-SOD was administrated into the third cerebral ventricle of unanesthetized, freely moving, ovariectomized (OVX) ewes. Jugular blood samples were taken every 15 min for 5 h before and 8 h after i.c.v. injections. In a pilot trial using three OVX ewes, i.c.v. injection of Cu,Zn-SOD at a dose of 0.5, 1.0 or 2.0 microg in 100 microl saline decreased plasma LH levels and abolished LH pulses, without affecting FSH secretion. In the main experiment, i.c.v. injection of 100 microl saline had no effect on mean LH levels and LH pulse frequency, whereas i.c.v. injection of Cu,Zn-SOD at a dose of 1 microg/100 microl saline significantly (P < 0.01) decreased mean LH levels and LH pulse frequency. In conclusion, this study provides the first evidence for the role of Cn,Zn-SOD in the control of LH secretion at the level of the brain in female mammals.

The conceptus regulates tryptophanyl-tRNA synthetase and superoxide dismutase 2 in the sheep caruncular endometrium during early pregnancy

Conceptus-derived paracrine signals play crucial roles in the preparation of a uterine environment capable of supporting implantation and development of the conceptus. However, little is known about the regulation of endometrial tryptophanyl tRNA synthetase (WARS) and manganese superoxide dismutase (SOD2) protein expression by the implanting and post-implanting conceptus. We hypothesized that the conceptus-derived signals favourably influences uterine environment for implantation through regulation of WARS and SOD2 expression in ovine caruncular endometrium. To test this hypothesis, WARS and SOD2 protein and mRNA expression was determined in caruncular endometrial tissues of unilaterally pregnant ewes at implantation (day 16) and post-implantation (day 20) periods. WARS protein expression increased in caruncular tissues of the gravid uterine horns compared with the non-gravid uterine horns on days 16 and 20 of pregnancy. There were no changes in SOD2 protein expression between the gravid and non-gravid uterine horns, irrespective of the day of pregnancy. On day 16 of pregnancy, there were no differences in WARS and SOD2 mRNA expression between the gravid and non-gravid uterine horns but expression of both genes was higher in the gravid uterine horns when compared with the non-gravid uterine horns on day 20 of pregnancy. In conclusion, the use of the unilaterally pregnant ewe model provides for the first time firm evidence that the early implantation and post-implanting conceptus-derived signals up-regulate WARS protein expression within the caruncular endometrium. Further studies are necessary to identify these signalling molecules and to understand mechanisms whereby they exert paracrine action within the endometrium.