The possible mitochondrial toxicity of sofosbuvir is underestimated and need intense investigations given in account the mass treatment by sofosbuvir at the national level like in Egypt. The present study aimed to assess the possible mitochondrial side effects of the preconception exposure of young female rats on the prenatal fetal tissues and placentas. The results raise serious concerns about the safety of sofosbuvir treatment in young female because it affects the pregnancy outcome and the mitochondrial biogenesis and function in fetal and placental tissue prenatally.
The preconception exposure of young female rats to Sofosbuvir results in significant decline in the pregnancy outcomes as indicated by lower numbers of viable embryos per letter and higher rats of fetal resorption (dead fetus). Also, fetal size and the fetal and placental weights are significantly higher than the control values. These effects may be related to the decreased quality of the oocytes due to the three months exposure to Sofosbuvir that may impairs the mitochondrial biogenesis and functions in the ovarian tissues of the mothers before the pregnancy which ultimately results in decreased fertility. This assumption is confirmed by our previous work indicated disturbed mitochondrial biogenesis and functions in the ovarian tissue due to treatment with Sofosbuvir (unpublished data). The low ovarian mtDNA-CN is a marker of poor oocyte quality, which may lead to poor embryonic development . The maternal mitochondrial dysfunctions and impaired biogenesis may be transmitted to following generations and may cause many diseases . Also, the possible direct effects of the Sofosbuvir administered before pregnancy cannot be ruled out since animal studies prove that Sofosbuvir metabolites cross the placenta and are excreted in the milk of nursing animals .
The placental defects may be another contributing factor for the decline in pregnancy outcomes and fetal abnormalities. The placenta is the first organ formed in pregnancy and it connects the maternal and embryonic circulations to facilitate the fetal supply with maternal nutrients . The placentomegaly observed in the pregnancies of Sofosbuvir-exposed mothers may indicate a state of chronic inflammation which confirmed in our study by induced placental expression of NF-κB. The placental inflammation may compromise its function as a selective barrier for the transport of nutrients and others into the fetus. In our study, placental inflammation was associated with significant increase in lipid peroxidation marker (MDA), and oxidized glutathione (GSSG) and significant drop in total and reduced GSH. These changes results in shifting the redox status from reducing environment (high GSH/GSSG ratio) into more oxidizing environment (low GSH/GSSG ratio). High concentrations of GSH are associated with a reducing environment and increased proliferation, while cell death is initiated by an oxidizing environment . Thus, the redox status of the placental tissues of Sofosbuvir-exposed mothers indicate a state of cell death and inflammation. The structural changes and maladaptation in the placenta may cause inflammatory shock to the fetus that may results in long-term adverse outcomes, including asthma, cerebral palsy, abnormal neurodevelopment, and autism spectrum disorder [21,22].
The observed changes in fetal and placental size and weight due to the maternal exposure to Sofosbuvir are associated with significant changes in the mitochondrial biogenesis and functions in fetal tissues and placentas and significant changes in the patterns of gene expression of genes controlling mitochondrial biogenesis and functions. However, the patterns of changes are not similar between the different organs and placenta.
Regarding the mitochondrial DNA copy number, an indicator of mitochondrial biogenesis, the placental tissues of Sofosbuvir-exposed mothers were increased significantly, the fetal liver showed no significant change, however the fetal muscle was declined significantly. The differential effects of the pre-conception maternal exposure to Sofosbuvir on the fetal tissues may explained by the fact that the different tissues are not homogeneous regarding the mtDNA-CN depending on the energy demand and the metabolic requirements. This explanation agrees with Pejznochova and colleagues who assumed that the non-homogenous distribution of mtDNA between fetal tissues is attributed to the differences in their metabolic roles, and that mtDNA-CN in fetal tissues is considered tissue-specific and depends on energy requirements as well as the stage of development . This explains the variation in mtDNA-CN in different sampled tissues in our study and the differential effects of
In the present study, the increased placental mtDNA copy number is associated with significant upregulation in the expression of PGC-1α and significant downregulation in the expression of POLG. The placental inflammation and oxidative stress may lead to mitochondrial proliferation to compensate the disrupted cellular bioenergetics. On the other hand, increased placental mitochondrial ROS may cause direct damage to mtDNA, thus, inhibiting mitochondrial biogenesis. However, in an attempt to maintain normal fetal growth, either increased or decreased mitochondrial biogenesis could take place .
Besides its key role as the main regulator of mitochondrial biogenesis, PGC-1α has been recognized as a powerful regulator of angiogenesis which is fundamental to the development of a healthy placenta in normal pregnancy. It may be possible that PGC-1α has a key role in the imbalance of angiogenic factors observed in pre-eclampsia. Delany and colleagues suggested that PGC-1α may be implicated in the pathogenesis of pre-eclampsia . Vishnyakova et al. found increased placental mitochondrial content in early-onset but not late-onset pre-eclampsia, suggesting that the different pathophysiology leads to differences in mitochondrial response . The maintenance of mitochondrial biogenesis during the critical period of fetal development is very crucial because the increased mitochondrial biogenesis during pregnancy is required to maintain metabolic activity of the placenta. Disruption of this process could increase the production of ROS and oxidative stress which subsequently leads to placental insufficiency and damage that is may be manifested as pre-eclampsia or intrauterine growth restriction .
In fetal muscle, the decline in mtDNA copy number is associated with marked induction of ND5 content and the expression of NRF-1 and POLG expression. These inductions may be an adaptive response to overcome the decline in mitochondrial biogenesis. Under normal physiological conditions, NRF-1 is widely expressed, and the highest expression has been recognized in skeletal muscles . Alone, NRF-1 was not enough to induce mitochondrial biogenesis, which was consistent with our findings. In this context, Baar et al., reported that the increased NRF-1 was not sufficient to initiate the expression of the proteins required for the assembly of functional mitochondria . Also, the mutations in POLG can cause either early childhood or later-onset syndromes resulting from mtDNA deletions .
Mitochondrial biogenesis is accompanied with skeletal myogenesis. Therefore, prenatal mitochondrial dysfunction and decreased mitochondrial biogenesis in skeletal muscles may manifest later in life, causing long-term consequences on energy homeostasis . Kang et al. found direct relation between Tfam and mtDNA, as heterozygous mutations of Tfam negatively affects mtDNA-CN, whereas homozygous mutation is embryonically lethal. Tfam heterozygous cells produce more inflammatory cytokines due to mitochondrial stress signaling. Hence, downregulation of Tfam expression resulted in mtDNA stress signaling. Levels of Tfam directly control mtDNA content, thus, any post-translational modifications that disrupt either the turnover or stability of Tfam could substantially impact the regulation of mtDNA abundance [23, 32].
In fetal liver, the pre-conception exposure of young females to sofosbovir contributed to mitochondrial dysfunction as indicated by significant decline in the content of ND5. NADH dehydrogenase, complex I, is the first and largest of the ETC complexes that has a pivotal role in energy metabolism as it is the main entry point for electrons to the ETC, hence, considered the rate-limiting step in overall respiration. Mutations in the genes encoding the complex I subunits have been associated with ETC disturbances and ROS production . Although the main pathway regulating mitochondrial biogenesis has been downregulated, mtDNA-CN, which represents a marker of mitochondrial biogenesis, does not significantly changed but showed mild decline.
In liver, mitochondria regulate lipid and amino acid metabolism, energy production and the urea cycle, as well as regulating the innate immune response to control . Sofosbuvir exposure causes fetal liver inflammation as shown by an increase in NF-KB expression, also increased oxidative stress as demonstrated by an elevation in MDA content and a decline in the GSH/GSSG ratio, which can contribute to the development and progression of liver diseases.
The observed prenatal effects of pre-conception maternal exposure to Sofosbuvir may have long-term adverse effects through impacting the health of the offspring in the adult life. The mechanism(s) of pre-conception Sofosbuvir exposure-induced changes in the pregnancy outcomes and fetal and placental tissues is (are) unclear and needs intensive study. However, we can suggest a different possible mechanism that may participate: (1) Sofosbuvir may directly or indirectly affect the quality of the ova through suppressing the gene expression of PGC-1α and/or Tfam that drives fetal abnormalities developed later after pregnancy, (2) Sofosbuvir may induce placental abnormalities and oxidative stress early in pregnancy that may affect the nutrient supply to the fetus which results in increased risk of mtDNA mutations, leading to mitochondrial dysfunction, and (3) Sofosbuvir may affect the hormonal homeostasis pre-conception and during pregnancy that may affect the fetal metabolism and development.