In this study the effects of MLT on NASH were assessed in an experimental model using C57BL/6 mice fed with a MCD diet, evaluating hepatic integrity, inflammatory processes, oxidative stress, and genotoxic/mutagenic effect markers. Marcolin et al. [18, 42] have shown that C57BL/6 mice, after 2 or 4 weeks fed with MCD, developed a NASH classical pathophysiological feature. Here, the animals were fed for 4 weeks and the treatment with MLT started 2 weeks after the beginning of the diets. NASH development was proved by evaluation of liver histopathological aspects, i. e., macrovesicular steatosis, hepatocellular ballooning, and fibrosis which already have been found in the previous studies [20, 42]. The treatment with MLT attenuated the steatosis, ballooning and fibrosis, indicating hepatoprotection.
Regarding AST and ALT, the levels of these enzymes were significantly elevated in the NASH group compared to the controls, while the MLT-treated group showed significantly lower levels compared to the NASH group, corroborating a previous study . Patients with NASH receiving MLT showed lower AST and ALT , however other studies did not find these enzymes altered by treatment with MLT [14, 17], indicating that other markers should be evaluated to assess MLT protection against steatohepatitis. In a study using English white pig with chronic steatohepatitis induced by a steatogenic diet, MLT was not able to decrease ALT, AST, and FA levels .
Pro-inflammatory cytokines are frequently increased in patients with NASH  and in experimental nonclinical models [19, 42] and might be used as liver markers in studies on steatohepatitis. In this study, the expression of TNF-α and iNOS increased in the NASH group, whereas in the MLT-treated group a significant reduction was observed, indicating an anti-inflammatory effect of MLT in this model, corroborating Tahan et al.  who have shown MLT decreased TNF-α, IL-1 β, and IL-6 using Wistar rats with steatohepatitis induced by MCD diet. Besides the inflammatory process, fibrosis is an important feature for the diagnosis of steatohepatitis, and it was shown by picrosirius staining and TGF-β expression which were increased in the NASH group. The presence of fibrosis in the hepatic tissue happens by accumulation of extracellular collagen due to the inflammatory process [43, 44]. In the NASH+MLT group there was a decrease in both hepatic fibrosis indicative parameters, showing that MLT inhibited the progression of liver damage. In another study, Bona et al.  have shown similar MLT action on pro-inflammatory markers and fibrosis (TGF-β) in a tetrachloride-induced cirrhosis model.
The accumulation of fat in hepatocytes stimulates Kupffer cells to release pro-inflammatory cytokines which exacerbate the formation of ROS, leading to oxidative stress . In this sense, the NASH group showed an increase in lipoperoxidation which also has been associated as a possible mediator to liver fibrosis because it influences collagen synthesis . In addition, GPx and SOD activities were reduced in the NASH group, while CAT increased, indicating a disbalance in antioxidant defenses due to oxidative stress triggered by hepatic steatosis. Previous studies have shown liver injuries due to oxidative stress in animals with NASH [19, 46, 47] corroborating this study. The treatment with MLT decreased lipoperoxidation and increased GPx and SOD while it decreased CAT, likely due to its antioxidant effects with the ability to directly scavenge ROS and reactive nitrogen species (RNS) and indirectly modulate antioxidant enzymes . In addition, MLT increased the Nrf2 expression which regulates the transcription of antioxidant responsive element (ARE) dependent genes to balance oxidative mediators and maintain cellular redox homeostasis [49–52]. Hence, the modulation of antioxidant enzyme activities as well as the decreased iNOS and TNF-α expressions were probably exacerbated by increased Nrf2 expression in mice with NASH treated with MLT, resulting in a decrease of oxidative stress.
Furthermore, the MLT treatment decreased DNA damage in blood and liver, likely due to its antioxidant and anti-inflammatory actions, diminishing ROS and RNS generation, avoiding the progression of NASH. Rezapoor et al.  have shown that MLT was able to increase the expression of genes related to DNA base excision repair (BER) such as Ogg1, Apex1, and Xrcc1 genes, which are associated with repair of free radical‑induced DNA damage. It could explain the lower DI and DF in blood and liver cells using Comet assay. In the liver, a direct scavenger effect by MLT and its metabolites such as cyclic 3-hydroxymelatonin, N1- acetyl-N 2-formyl-5-methoxyquinuramine (AFMK) and N1- acetyl-5-methoxyquinuramine (AMK) might have contributed to protective effects on DNA, besides the indirect actions by increasing the DNA repair capacity. As observed in this study, MLT was able to decrease the expression of TNF-α and TGF-β pro-inflammatory cytokines and iNOS, modulate antioxidant activities regarding CAT, SOD, and GPx and increase the expression of the Nrf-2, creating an adequate anti-inflammatory environment to protect the genetic material as well as decreasing lipoperoxidation. This is the first study showing the ability of MLT to avoid DNA strand breaks in blood and liver cells of mice with NASH. In cirrhotic rats, MLT also decreased DNA damage in liver [23, 54]. Other compounds such as quercetin and simvastatin have shown similar responses in mice with NASH induced by the MCD diet, decreasing DNA damage in liver [20, 26].
DNA damage in blood was also observed in NASH mice, showing that the inflammation processes elicited in NASH might affect other tissues besides liver, although no increase in MNPCE in the NASH group was observed, showing that the bone marrow cells were not affected by the systemic injuries triggered by the disease. Conversely, in our previous studies, cirrhotic rats have not shown an increase in DNA damage in blood cells, but the MNPCE frequency increased [23–25]. These findings suggest that in the course of progression from NASH to cirrhosis the damaged blood cells could be removed or repaired, however the higher liver injuries in cirrhotic rats in comparison to NASH lead to systemic repercussions able to increase the genomic instability detected using the micronucleus test. It is known that, in the progression of hepatic diseases, other organs are gradually impaired, such as lung and skeletal muscles besides the liver, especially by inflammatory processes [24, 29, 54, 55].
Micronuclei are produced in dividing cells that contain breaks in chromosomes without centromeres and/or whole chromosomes which are unable to migrate to the poles during mitosis, and their presence in recently divided cells indicates genomic damage without the possibility of repair [56, 57]. The chemical or physical clastogenic/aneugenic agents might increase the micronucleus frequency and are considered mutagenic agents. Furthermore, the association between aging and the reduction of antioxidant defenses and the increase in DNA repair deficiencies leading to higher MNPCE frequency is well known . In a study to evaluate MLT on aging, Damiani et al.  have observed that MLT supplementation time is associated with DNA damage and micronucleus frequency that are lower in Swiss mice. Similarly, diseases involving an inflammatory process might increase the genomic instability detectable using the micronucleus test [21, 59, 60]. As already mentioned, no genomic instability was detected in the NASH model induced-MCD diet under conditions of the present study using micronucleus test in bone marrow, differently from other liver injury models such as cirrhosis induced by secondary bile duct ligation model [23–25], suggesting that genomic instability increases according to the progression of liver disease stages.
Since MNPCE frequency did not increase in the NASH group, no antimutagenic activity could be observed in the treatment with MLT. In a study to evaluate MLT protective effects against genotoxicity induced by ethanol in pregnant mice, antigenotoxic effects were shown, decreasing DNA damage in blood and liver. However, no antimutagenic activity was shown using the micronucleus test in bone marrow . Other studies have shown antimutagenic effects of MLT such as reducing MNPCE frequency in mice treated with paraquat, a herbicide that generates free radicals . Melatonin was also able to decrease DNA damage to blood, liver, and brain and micronucleus induced by exposure to pesticide cypermethrin in rat dams and offspring . It decreased DNA damage in blood and micronucleus frequency in bone marrow of mice exposed to formaldehyde by inhalation . Melatonin has prevented diabetes-related DNA damage of hepatocytes of rats treated for 4 weeks with low doses (0.2 mg/kg at MTL) . In addition, melatonin has shown anti-cancer [66, 67] and protective effects against carcinogens such as dimethyl bez (a) anthracene and diethylnitrosamine, reducing DNA damage evaluated using Comet assay in blood and liver cells of treated rats [34, 68], indicating its capacity to protect the genetic material.