Epidemiological data on hypermethioninemia are extremely limited; however, Villani et
al. (2019) demonstrated in a ten-year study that two of seventy-seven thousand newborns were diagnosed with hypermethioninemia: one with classic homocystinuria and the other with MAT I/III deficiency (Villani et al., 2019). Martins et al. (2012) demonstrated an incidence of one out of twenty-six thousand newborns with the condition on an island in Portugal (Martins et al., 2012), while Marcão et al. (2015) demonstrated a frequency of approximately one out of twenty-seven thousand four hundred cases of MAT I/III deficiency (Marcão et al., 2015).
It is known that the dominant form is typically benign, but the recessive form of the disease can cause several serious neurological and hepatic alterations, in addition to changes in clinical signs and symptoms, as demonstrated in several studies (Lu & Mato, 2008; Lu et al., 2001; Allen et al., 2019; Kido et al., 2019; Nashabat et al., 2018; Zhang et al., 2020; Sem et al., 2019). However, the pathophysiological mechanisms involved in these symptoms are poorly understood. We investigated the possible involvement of changes in redox status in the brain, liver, and kidney in a recently standardized model of hypermethioninemia in young mice. The plasma concentrations of Met, 1 h after administration of Met, MetO, and Met + MetO, were similar in the mice to those found in patients with isolated and persistent hypermethioninemia (Franceschi et al., 2020).
We demonstrated that there was an increase in the production of ROS in the brain in all experimental groups, which could be caused by a change in mitochondrial activity, associated with a reduction in the activity of the detoxifying enzymes SOD and CAT. Accordingly, Soares et al. (2017) demonstrated that in an acute protocol, Met and MetO reduce the mitochondrial electrochemical potential. A decrease in antioxidant enzyme activity has also been reported previously in macrophages of mice in vivo and in vitro (Franceschi et al., 2020; Dos Santos et al., 2017).
An increase in ROS levels can cause a series of deleterious events in the central nervous system. Oxidative damage in the brain may have occurred due to the increased production of ROS, reduced antioxidant activity, and the high levels of membrane polyunsaturated lipids, which can be easily oxidized, leading to lipid peroxidation, which is assessed by TBARS levels. Altogether, these changes can modify cell homeostasis and lead to cell death, as reported by Soares et al. (2017). Thus, it can be suggested that oxidative damage may be involved in the pathophysiology of neurological damage found in patients with hypermethioninemia, since this condition can lead to damage to other important biomolecules such as proteins and DNA.
Considering that the liver and kidneys are very important for the metabolism of amino acids and that liver changes have already been reported in patients with hypermethioninemia (Lu & Mato, 2008; Lu et al., 2001), it is worthwhile to investigate the effects of chronic administration of Met and/or MetO on oxidative markers in these tissues.
In the present study, Met and/or MetO increased the liver production of ROS and nitrite. In addition to mitochondria, the endoplasmic reticulum can also be a significant source of ROS via cytochrome P450 enzymes. Additionally, when compared to the control, the reduction of activity of SOD and CAT, observed in all experimental groups, can contribute significantly to the accumulation of reactive species. It is important to note that reducing SOD activity concomitantly with increasing nitrite level may favor the production of peroxynitrite, which is a reactive species that is very harmful to biomolecules. However, as the liver is a central organ of metabolism, it is rich in enzymatic and non-enzymatic antioxidant defenses (Czaja, 2007; Cichoż-Lach and Michalak, 2014); therefore, an imbalance that is capable of altering liver redox homeostasis should be considered with caution.
In general, chronic diseases of hepatic origin are characterized by the exacerbated presence of oxidative stress, regardless of the cause of the disease (Czaja, 2007; Cichoż-Lach and Michalak, 2014). In this context, some studies have reported that the expression of certain proteins can be modulated by free radicals, and that this modulation can occur in relation to the activation of redox-sensitive transcription factors, such as Egr-1, NF-kappaB and AP-1, as well as G proteins. Cellular kinases are a mitogen-activated protein kinase family that also plays an essential role (Czaja, 2007; Cichoż-Lach and Michalak, 2014). Considering the power of oxidative stress in modulating important signaling pathways for cellular homeostasis, the identification of this condition in hypermethioninemia is extremely relevant, as it may contribute significantly to the liver damage caused by cirrhosis and steatosis, already reported in hypermethioninemic patients (Lu & Mato, 2008; Lu et al., 2001).
A reduction in ALA-D enzyme activity in the liver was also found in groups treated with Met and/or MetO. The ALA-D enzyme catalyzes the second reaction in the heme biosynthesis pathway and is expressed in all tissues, but is more common in the liver. In contrast to the results of Soares et al. (2017), a reduction in the activity of ALA-D was observed, and this can lead to the development of signs and symptoms of typical hepatic porphyria, a condition that is detected in other inborn errors of metabolism such as type 1 tyrosinemia (Fujita et al., 1995; Kelada et al., 2001). ALA-D activity can be reduced in the presence of oxidizing agents, such as free radicals, and this reduction can lead to pathological consequences with damage to the heme biosynthesis route, accumulation of its substrate in the blood, and overproduction of ROS (Fujita et al., 1995; Kelada et al., 2001).
Finally, we also demonstrated that Met and/or MetO induces an increase in the production of ROS, a reduction in the levels of total thiol content, and a reduction in CAT activity. In contrast, SOD activity was significantly increased in the MetO and Met + MetO groups. The increase in SOD activity can be a compensatory mechanism for the high ROS production. However, it can be postulated that even with increased SOD enzyme activity, damage to the SH groups still occurs. Furthermore, knowing that SOD is responsible for the dismutation of the superoxide anion to hydrogen peroxide, another possible conclusion is that oxidative damage in the kidney is caused by the accumulation of hydrogen peroxide, since there is an increased production of this substance, and a reduction in detoxification caused by CAT inhibition.
The data found in the present study corroborate and complement the previous findings of the literature demonstrating that high doses of Met and/or MetO, similar to those found in patients with hypermethioninemia, can be extremely harmful to several organs, particularly the brain, liver, and kidney (Soares 2020b; Soares et al., 2017; Stefanello et al., 2009). In addition, it should be noted that one of the possible intensifiers of this damage is the presence of MetO, since this metabolite can not only cause damage itself, but is able to form other metabolites with even greater toxic potential, such as methionine sulfone and homocysteic acid. Another interesting point is that the oxidative damage caused by experimental hypermethioninemia in isolated brain structures remains when the total brain is evaluated. Finally, the importance of investigating the possible pathophysiological mechanisms of hypermethioninemia in several experimental models is highlighted, with the purposes of expanding our knowledge on this pathology and determining how high doses of amino acids act in different conditions and biological models.