Constant light exposure and/or pinealectomy increases susceptibility to trichloroethylene-induced hepatotoxicity and liver cancer in male mice

Exposure to light at night, pineal gland impairment, and the environmental pollutant trichloroethylene (TCE) have serious implications for health and contribute to illness, including liver cancer. The adverse effect of the association of continuous exposure to light with decreased melatonin levels and TCE-induced toxicity is not disclosed in target organs. This work explored the role of light and pineal impairment in increasing susceptibility to liver toxicity and cancer upon exposure to TCE. Male albino mice were divided into groups as follows: control group (12-h light/12-h dark cycle), constant light (24-h light), pinealectomized (Pnx) mice, sham surgically treated group, TCE-treated groups subjected to two doses (500 and 1000 mg/kg) at two different light regimens, and combination of Pnx and TCE-treated mice kept at a 12-h light/12-h dark cycle. Melatonin levels were significantly decreased in both Pnx mice and TCE-treated animals at both light regimens. Aspartate transaminase, alanine aminotransferase, activities, and serum bilirubin levels were significantly elevated, whereas albumin levels were markedly decreased in Pnx mice, TCE-treated mice, and the combination group. Histopathological investigations reflected changes in liver function parameters indicating liver injury and induction of cancer. These effects were accompanied by significant increase of the liver cancer biomarker alpha-fetoprotein and the expression of the metastatic markers CD44, TGFβ-1, and VEGF, along with increased oxidative stress indicators and inflammatory cytokines (IL-6, IL-1β, and TNF-α) in both Pnx and TCE-treated mice and the combination group at both light regimens. Taken together, our findings indicated that low melatonin levels, exposure to constant light, and the combination of both factors increases susceptibility to the toxic and carcinogenic effects of TCE on the liver.


Introduction
Light is a crucial physiological influencer of circadian organization of the organs of the human body and daily activity. Shift work and changes in sleep-wake behavior that increase exposure to light at night are prevalent in most modern societies. People in both categories display disruption in melatonin production, and decreased levels of melatonin release into the blood at night could increase the risk of several disorders and diseases, including cancer (Walker et al. 2020). All mammals possess a circadian timing system that generates 24-h rhythms in many physiological processes (van Zuylen et al. 2021). The rodent and human circadian timing system share many common features.
Melatonin (N-acetyl-5-methoxytryptamine) is a neurohormone synthesized and released primarily by the pineal gland. Melatonin production is enhanced at night and reduced by light at daytime (Zisapel 2018). Disturbance of circadian rhythm can be a result of exposure to artificial light during night time and decreased melatonin synthesis and release due to pineal dysfunction (Burgess and Emens 2020). Night shift workers are highly susceptible to cancer (Touitou et al. 2017). Suppression of melatonin by light during night shift work and due to pineal impairment have remarkable influence on cancer development (Hunter and Figueiro 2017). This is attributed to decreased melatonin levels and disturbed circadian rhythm due to exposure to artificial light at night (Hunter and Figueiro 2017). A number of studies have shown that decreased melatonin levels are associated with the development of several cancers (Carter et al. 2014;Manouchehri et al. 2021;Schernhammer et al. 2003). On the other hand, highly credible evidence reports the anticancer effects of melatonin (Reiter et al. 2017). Several studies have documented the preventive and therapeutic effects of melatonin on the initiation and development of several cancers, which are attributed to its potent antioxidant and free radical scavenging activity (Amin et al. 2019;Reiter et al. 2017;Talib 2018). Additionally, outdoor blue light that is prevalent in recent years has been shown to suppress melatonin and increase cancer risk (Garcia-Saenz et al. 2020). An association between average daily television viewing time and the incidence of ovarian cancer has also been reported (Ukawa et al. 2018). Additionally, increased cancer risk is associated with short sleep duration in Asian populations .
Trichloroethylene (TCE) is an environmental and occupational contaminant that is produced and applied on a large scale and often disposed inappropriately (Horzmann et al. 2020). The primary sources releasing TCE into the environment are metal cleaning and degreasing operations (Wu and Schaum 2000). It is a well-known carcinogen and is associated with several toxic effects, including immunotoxicity, reproductive toxicity, neurotoxicity, cardiotoxicity (Huang et al. 2021), and teratogenic effects (Huang et al. 2015), and is a potential risk factor in the development of neurodegenerative disorders (De Miranda and Greenamyre 2020). Despite the associations of TCE exposure with many cancers and diseases, the molecular mechanisms of TCE-induced negative health effects are unclear. A recent study reported on the association between TCE exposure and increased DNA methylation of genes encoding markers of autoimmune disease and cancer (Phillips et al. 2019). Pathomechanisms of toxicity include dysfunction of mitochondria and disrupted membrane potential (Elkin et al. 2019), oxidative stress (Elkin et al. 2018), and excessive production of proinflammatory cytokines (Hassan et al. 2016). The effect of low melatonin level and/or constant light exposure on susceptibility of TCE-induced liver cancer development is unclear.
To the best of our knowledge, there have been no studies on the association of continuous exposure to light with decreased melatonin levels and TCE-induced toxicity. This work aimed to evaluate the role of light at night and pineal impairment in increasing the hepatotoxicity and incidence of liver cancer development upon exposure to the environment pollutant TCE.

Chemicals
Melatonin and TCE were obtained from Sigma Chemical Company, USA. All other chemicals were of highest analytical grade and obtained from Al-Gomhoria Chemical Company, Egypt.

Animals
Adult male albino mice weighing 25-30 g were obtained from the Egyptian Vaccine Company (VACSERA, Giza, Egypt). During the experimental period, mice were given a standard rodent diet, water ad libitum, and maintained at room temperature for a week before the experiment. The experimental protocol for the treatment of mice was performed according to guidelines and approved by the Institutional Animal Ethics of Mansoura University committee (Sc-Z-M-2021-42).

Animal groups and experimental design
Animals were divided into ten groups, 5 mice each, as follows: Group 1 (Control 12-h L): In which mice did not receive any treatment except the vehicle (corn oil) and exposed to 12-h light (L)/12-h dark (D) cycle. Light intensity of 175-200 lx was used. During accommodation and experimentation, light was emitted by white ceiling fluorescent tubes along the middle of the room. The cages were positioned in a manner to ensure medium light intensity average of 175 lx. Light intensity was verified in the animal house with a light meter (Light meter UNI-T UT383S, China). The experimental period was 30 days. Group 2 (Control 24-h L): Mice maintained at 24-h L in similar conditions as the Group 1 control group for 30 days at light intensity 175-200 lx. Group 3 (Sham): Mice subjected to surgery without removal of the pineal gland. These mice were kept at 12-h L/12-h D cycle, and the light intensity was 175-200 lx during the experimental period. Group 4 (Pnx 12-h L): In which the pineal glands were removed surgically as previously described (Maganhin et al. 2009). These mice were kept at 12-h L/12-h D cycle, and the light intensity was 175-200 lx. Group 5 (TCE 500 mg, 12-h L): Mice exposed to 12-h L/12-h D cycle and given TCE 500 mg/kg daily by stomach tube for 6 days, then left without treatment till the end of experimental period. TCE was prepared in corn oil. Group 6 (TCE 500 mg, 24-h L): Mice exposed to 24-h L and treated with TCE 500 mg/kg daily for 6 days, which was continued till the end of the experimental period of 30 days in constant light. Group 7 (Pnx + TCE 500 mg, 12-h L): Pinealectomized animals exposed to 12-h L/12-h D cycle and treated with TCE 500 mg/kg daily for 6 days in a 12-h L/12-h D cycle. Group 8 (TCE 1000 mg, 12 h L): Animals exposed to 12-h L/12-h D cycle and treated with TCE 1000 mg/kg daily for 6 days and kept at a 12-h L/12-h D light cycle for 30 days. Group 9 (TCE 1000 mg, 24-h L): Mice exposed to 24-h light cycle and treated with TCE 1000 mg/kg daily for 6 days in constant light. Group 10 (Pnx + TCE 1000 mg, 12 h L): Pinealectomized animals exposed to a 12-h L/12-h D cycle and treated with TCE 10,000 mg/kg daily for 6 days.

Surgical procedure for pinealectomy
Pinealectomy was performed as previously described (Maganhin et al. 2009). Briefly, overnight fasted mice were anesthetized with 15-mg/kg xylazine and 30-mg/kg ketamine, then subjected to a longitudinal opening in the scalp to expose the lambda suture. The skull around the lambda suture was carefully removed, followed by the pineal gland removal using fine forceps. The cranium bone was placed back to its original position, and the scalp was sutured. The procedure was completed within 30 min. After surgery, the animals received a single prophylactic antibiotic (amoxicillin) and analgesic (ketoprofen) via the intramuscular route. All operated mice were allowed to recover for 2 weeks before starting the experiment.

Sample collection
The experiment lasted for 30 days, after which the animals were euthanized with ketamine/xylazine (0.1 ml/100 g, i.p.) for blood and liver collection.
Blood samples were collected directly from hearts into clean tubes. Sera were prepared by centrifugation at 1500 × g and then used for biochemical assays. The mice were dissected, and livers were harvested. Portions of the liver were homogenized in cold phosphate buffer, centrifuged at 3000 × g to obtain the supernatant, and kept at − 4 °C for biochemical analysis. Other liver portions were fixed for 48 h in 10% buffered neutral formalin (pH 7.4) for histological and immunohistochemical investigation.

Determination of biochemical parameters in serum
A melatonin ELISA kit obtained from Fine Test, EM1218 Wuhan Biotech, China, was used to determine free melatonin serum levels according to the manufacturer's instructions. Melatonin levels were expressed as pg/ml. Alfa fetoprotein (AFP) serum levels were also determined through ELISA in accordance with the manufacturer's instructions using kits obtained from Bio-Techne (MAFP00) Minneapolis, USA. The level of vascular endothelial growth factor (VEGF) in serum was estimated using an ELISA kit (EK0541) obtained from BosterBio, USA.
Cytokines levels were estimated by ELISA kits obtained from My Biosource (San Diego, USA) according to the instruction manual for interleukin (IL)-10 (MBS824703), IL1-β (MBS175967), and IL-10 (MBS704754). Levels of tumor necrosis factor alpha (TNF-a) were evaluated by an ELISA kit (ELM-TNFa) obtained from RayBiotech, GA, USA. Liver function parameters, namely, aspartate transaminase (AST), alanine aminotransferase (ALT), albumin, and bilirubin, were determined calorimetrically in serum according to the instruction manual of the kits obtained from Biodiagnostics, Giza, Egypt. The levels of glutathione (GSH) and activities of glutathione peroxidase (GPx) and glutathione reductase (GR) in the liver were determined in accordance with the instructions in the manual of kits obtained from Biodiagnostics, Egypt. The levels of malondialdehyde (MDA), hydrogen peroxide (H 2 O 2 ), and nitric oxide (NO) were determined calorimetrically in liver following the instruction of the kit purchased from Biodiagnostics, Giza, Egypt.

Histopathology
A standard procedure was adopted to prepare paraffin wax blocks containing liver samples; then, 5-μm sections were prepared. The sections were processed following the standard procedure for hematoxylin and eosin staining for histopathological observation. Stained sections were then observed using an Olympus light microscope and photographed using an Amscope MU1000 camera. The extent of hepatic tissue injury was then evaluated via semiquantitative scoring in five randomly selected fields for each section. The scoring system relied on the tissue involvement percentage, as described (Khafaga and El-Sayed 2018), considering hepatic cords arrangement, sinusoidal dilation and hepatocytic necrosis. Liver injury parameters were scored as none (0): no involvement of evaluated field; mild (1): involvement of 0-25% of evaluated field; moderate (2): involvement of 25-50% of evaluated field; and severe (3): involvement of 50-100% of evaluated field (Khafaga et al. 2019).

Statistical analysis
Data were analyzed using the statistical software program Prism (GraphPad, Prism, 6.01). The mean ± standard deviation of each variable was estimated. Two-way ANOVA test was used to compare effect of combined independent factors on single continuous parametric outcome with post hoc Tukey test for pairwise comparison. Differences were considered statistically significant at P < 0.05.

Comparison of melatonin levels across study groups
The serum melatonin levels of all animal groups were evaluated and are presented in Fig. 1. The mice exposed to 24-h L showed a significant (P < 0.05) decrease in the serum melatonin compared with the control group exposed to the 12-h L/12-h D cycle. The Pnx mice exposed to 12-h L/12-h D cycle exhibited a significant (P < 0.05) decrease in melatonin levels in blood. The treatment with either 500 or 1000 mg/kg TCE caused a significant (P < 0.05) decrease in melatonin levels in animals exposed to 12-h L/12-h D and 24-h L compared with the control animals. The Pnx mice that received both doses of TCE also exhibited a significant (P < 0.05) decrease in blood melatonin compared with the control animals. The sham surgery group showed an insignificant change from the control value.

Serum levels of liver function parameters, AST, ALT, albumin, and bilirubin
We determined expression changes in liver function parameters, including the activity of AST and ALT and levels of albumin and bilirubin in serum, across the study groups (Fig. 2). The data showed an insignificant change in these parameters in serum of mice kept at constant light and shamoperated mice compared with the control (12-h L/12-h D cycle) mice. On the other hand, Pnx mice and TCE-treated mice kept in both light regimens showed a significant (P < 0.05) elevation in the activity of ALT and AST and serum bilirubin. Additionally, the albumin concentration in the serum of these rats showed a significant (P < 0.05) decrease compared with control animals kept in 12-h L/12-h D cycle. Pnx mice treated with both doses of TCE showed higher values compared with rats treated with TCE at both light regimens.

Liver histopathology findings
The livers of 12-h L/12-h D-exposed animals showed normal hepatocytes arranged in cords around the central vein (grade 0 injury) (Fig. 3A, B). On the other hand, the livers of 24-h L-exposed mice showed moderate alteration in the structural Values are expressed as mean ± standard deviation of the mean. Same letters represent non-significant difference between groups. Differences were considered statistically significant at P < 0.05 organization of the hepatic lobules, hemorrhage, and mild dilation of blood sinusoids. Additionally, mild degree of cellular atypia within hepatocytes was observed (Fig. 3A, B).
The livers of mice subjected to 12-h L/12-h D and treated with TCE 500 mg/kg and the group that exposed to constant light showed moderate degree of cellular atypia within hepatic cells. Sections from the livers of TCE 500 mg, 24-h L mice showed liver damage exhibited by loss of liver architecture, dilation of blood sinusoids, and cellular atypia of eosinophilic foci of affected hepatocytes (grade 5 injury) (Fig. 3A, B).
Pnx mice subjected to 12-h L exhibited marked alteration in liver architecture with diffused hepatic granular vacuolation as well as hemorrhage (grade 4 injury) (Fig. 3A, B). In Pnx, TCE 500 mg, and 12-h L mice, the liver sections displayed clear preneoplastic foci associated with tigrolysis of hepatocytic cytoplasm. This was accompanied by dilation of blood sinusoids with hemorrhage (grade 6 injury). The Pnx, TCE 1000 mg, and 12-h L treated group showed complete deterioration of liver architecture, with massive dilation of both blood sinusoids and central vein with hemorrhage, pyknosis, and ballooning degeneration of hepatocytes as well as leukocytic infiltration. These changes are associated with clear preneoplastic foci that compress the hepatic parenchyma (grade 8 injury). mean ± standard deviation of the mean. Same letters represent nonsignificant difference between groups. Differences were considered statistically significant at P < 0.05

Serum expression levels of AFP and VEGF
There were no changes in AFP serum levels of mice kept in constant light compared with the control (12-h L/12-h D cycle). On the other hand, pinealectomized rats showed a significant (P < 0.05) increase in serum AFP compared with control animals. Similarly, all animals treated with both doses of TCE and liver in both light regimens as well as Pnx mice that were treated with TCE and kept in 12-h L displayed a significant (P < 0.001) increase in serum AFP levels compared with the control groups. The latter group showed higher values of AFP than other TCE-treated groups. The angiogenic factor VEGF was evaluated in serum. The level of VEGF was insignificantly changed in animals exposed to 24-h light compared with the control group (12-h L/12-h D cycle). Pnx mice kept in the normal light-dark cycle showed a significant (P < 0.05) increase in levels of VEGF in serum.
Similarly, treatment with either dose of TCE caused a significant (P < 0.001) increase in VEGF in serum of all animals subjected to 24-h L than the control. Pinealectomy and treatment with TCE showed a significant (P < 0.05) increase in the serum level of VEGF compared with other TCE-only treated and the control groups (Fig. 4).

Immunohistochemical evaluation of CD44 expression
CD44 is a cancer cell metastasis cell surface protein marker. Therefore, we immunohistochemically determined CD44 expression in mice liver and compared expression levels across the study groups (Fig. 5). A significant increase in the expression of CD44 was observed for all experimental groups compared with control group (12-h L/12-h D cycle). Pinealectomy further elevated the number of CD44-labeled  Figure 6 shows transforming growth factor beta-1 (TGFβ-1) expression in liver cells. TGFβ-1 expression significantly (P < 0.05) increased in all pinealectomized mice as well as TCE-treated groups at both light regimens. Combination of pinealectomy and treatment with TCE showed higher values of TGFβ-1 compared with other groups.

Liver antioxidant expression levels
We observed a significant decrease in antioxidant enzyme activities (SOD, CAT, GPx, and GR), and GSH deviation of the mean. Same letters represent non-significant difference between groups. Differences were considered statistically significant at P < 0.05 concentration in liver of mice kept at 24-h L compared with control mice 12-h L/12-h D (Fig. 7). The Pnx mice maintained 12-h L/12-h D cycle showed a significant (P < 0.05) decrease in liver antioxidant levels. There was a significant (P < 0.001) decrease in antioxidants in all animals treated with TCE compared with the control mice (Fig. 7). The Pnx mice treated with TCE and maintained in 12-h L/12-h D showed remarkable decrease (P < 0.05) in liver antioxidants deviation of 3 microscopic fields/tissue samples. Same letters represent non-significant difference between groups. Differences were considered statistically significant at P < 0.05 compared with animals treated only with both doses of TCE and subjected to same light regimen.

Oxidative stress marker expression profiles
Mice exposed to 24-h L exhibited an insignificant change in the oxidative stress markers (H 2 O 2 , MDA, PC, and NO) in liver compared with the control animals (12-h L/12D cycle) (Fig. 8). The Pnx mice maintained 12-h L/12-h D showed a significant (P < 0.05) increase in investigated oxidative stress markers. Treatment of animals with either dose of TCE produced a significant (P < 0.05) increase in H 2 O 2 , PC, and NO in 24-h L compared with the control mice (12-h L/12-h D). The MDA level showed an insignificant change in the same animals. The Pnx mice treated with TCE and maintained in 12-h L/12-h D showed a remarkable increase (P < 0.05) in oxidative markers in the liver compared with animals treated with TCE only with either dose and subjected to the same light regimen (Fig. 8).

Proinflammatory mediator expression changes
Mice exposed to 24-h L exhibited an insignificant change in the proinflammatory cytokine (IL-6, IL-1β, and TNFα) expression. However, a significant decrease (P < 0.05) in IL-10 was observed in 24-h L compared with control (12-h L/12-D cycle) (Fig. 9). Animals subjected to pinealectomy showed a significant increase in the proinflammatory cytokines and a significant (P < 0.05) decrease in IL-10. Treatment of animals with either dose of TCE produced a significant (P < 0.05) increase in IL-6, IL-1β, and TNF-α and a significant decrease in IL-10 compared with the control group (Fig. 9). The effect of TCE on the pro-and anti-inflammatory cytokines was more evident in Pnx and TCE-treated mice kept at constant light than in the control animals.

Discussion
The aim of the present study was to determine the effect of constant light exposure, and pinealectomy on susceptibility to TCE-induced liver toxicity and cancer induction. Our findings suggest that constant light and pinealectomy increase the TCE-induced hepatotoxic effect characterized by expression of cancer markers through upregulation of oxidative stress and inflammation. The findings of this study showed a significant decrease in melatonin levels in the blood of animals subjected to 24-h light or pinealectomy for 30 days compared with the control group (12-h L/12-h D). Exposure to light at night, the time for elevated Values are expressed as mean ± standard deviation of the mean. Same letters represent non-significant difference between groups. Differences were considered statistically significant at P < 0.05 expression of melatonin, has been reported to inhibit melatonin synthesis and secretion (Touitou et al. 2017). Exposure to artificial light at night is a source of pollution and a growing public health issue in modern societies. This phenomenon is referred to as light stress because it affects the circadian clock (Touitou and Point 2020) and induces sleep deprivation. The present study revealed that removal of the pineal gland resulted in a significant decrease in serum melatonin levels with increased oxidative stress in the liver and inflammatory cytokines in blood. The increased level of H 2 O 2 and NO augmented protein oxidation in mice liver. The elevated levels of oxidative markers are attributed to a remarkable decrease in enzymatic (SOD, CAT, GPx, and GR) and non-enzymatic antioxidants (GSH and melatonin) in liver of animals kept in constant light and/or exhibiting pineal impairment (Cichoż-Lach and Michalak 2014). These findings suggest that exposure to constant illumination at night and/or pinealectomy stimulates redox state and the undesirable influences of oxidative radicals.
Oxidative stress is the main causative factor for several liver diseases . The activated redox state influences pathways of inflammatory, metabolic, and proliferative liver diseases (Cichoż-Lach and Michalak 2014). Melatonin is the main antioxidant and free radical scavenger system in the body that clears the buildup of free radicals during the day, mitigating oxidative stress and inflammatory responses and enhancing anti-inflammatory impact under several conditions exposure to ionizing radiation and diabetes (El-Missiry et al. 2020, 2007. It was recently reported that constant light not only eliminated the circadian rhythms of the expression of the clock genes but also eliminated the circadian rhythms in the genes involved in lipid metabolism in liver and fat cells (Yamamuro et al. 2020). Because circadian rhythm depends on an internal clock under the control of the pineal gland and melatonin level in the blood (Aulinas 2000), it is suggested that constant light and pineal gland impairment are strong chronodisruptors affecting physiological function of the body's organs, including the liver. The present study showed that pinealectomy or exposure to constant light resulted in a significant increase in AFP, CD44, TGFβ-1, and VEGF levels indicating early development and progression of hepatic cancer cells. These data were further confirmed by histopathological examination of Pnx and TCE-treated groups, which demonstrated preneoplastic foci and ballooned cells, which are associated with tigrolysis of the hepatic cytoplasm and nuclear atypia. AFP levels have been reported to significantly elevate in liver damage and cancers, and thus, has been used as a tumor marker (Jiang et al. 2018). Emerging evidence has illustrated the role of ROS in stimulating the expression of TGFβ-1, which in turn, in a positive feedback loop, increases ROS generation by diminishing antioxidant enzyme expression (Liu and Desai 2015). The involvement of TGFβ-1 in signaling for cancer occurrence and development is illustrated in several studies (Liu et al. 2019). Meanwhile, melatonin in a dose-dependent manner significantly attenuated the TGFβ-1-dependent stimulation of epithelial-mesenchymal transition in mouse AML12 hepatocyte cells by deactivating ROS signaling (Kim et al. 2019). These data suggest that melatonin is crucial factor for protection against liver cancer development through controlling signaling pathways. While melatonin treatment inhibits tumor growth, metabolism, and proliferation, pinealectomy and/or constant light exposure stimulates tumor growth (Blask et al. 1999). Treatment with melatonin inhibits mammary carcinogenesis in pinealectomized rat kept in the standard light/dark regimen or under constant illumination regimen (Anisimov 2003). Thus, pinealectomy or constant light exposure stimulate adverse health problems and induce cancer development. Therefore, we suggest that light and melatonin are environmental signals that collaborate with each other to control body physiology and regulate tumor initiation and cancer induction. It is also suggested that elevation in oxidative stress and inflammatory cytokine levels due to low melatonin levels might contribute to upregulation of tumor markers in the liver.
It is believed that cancer incidence increased with increased exposure to TCE (Wartenberg et al. 2000). In the present study, TCE exhibited a significant carcinogenic effect in normal (12-h L/12-h D), constant light (24-h L) exposed rats and in pinealectomized rats as well as in pinealectomized rats subjected are expressed as mean ± standard deviation of the mean. Same letters represent non-significant difference between groups. Differences were considered statistically significant at P < 0.05 to TCE compared with normal rats kept in 12-h L/12-h D cycle. The present study demonstrated a severe reduction in melatonin levels with a significant increase in AFP, CD44, TGFβ-1, and VEGF as well as proinflammatory cytokines, including IL-6, IL-1β and TNF-α, with marked decrease in the anti-inflammatory cytokine (IL-10) in these animals. It is reported that TCE increases the differentiation of Th17 cells and increases IL-17 secretion by inducing IL-6 with TGF-β (Shen et al. 2012). The decreased melatonin level after TCE treatment might indicate pineal dysfunction. The combination of pinealectomy and TCE displayed the highest effect on these parameters, which might be attributed to low melatonin levels and increased oxidative stress. TCE and constant light exposure induced remarkable reduction in melatonin levels and antioxidants, with an elevation in oxidants that lead to increased oxidative stress in the liver. Oxidative stress plays a primary role in the starting action of hepatic and extrahepatic damage (Masarone et al. 2018). These results suggest that constant light and pineal ectomy increased mice susceptibility to TCE-induced toxicity and promoted cancer. TCE and its metabolites can produce liver cancer in mice and put humans at risk of developing liver cancer through several mechanisms, including somatic mutation and modification of cell signaling pathways, but the actual mechanisms involved have not been established (Bull 2000). TCE significantly increases oxidative stress and inflammatory cytokines, with a decline in antioxidant protection and antiinflammatory (IL-10) levels. These effects are very likely attributable to low melatonin levels. The toxic effect of TCE was more pronounced when combined with constant light and was exuberated by pinealectomy than in the 12-h L/12h D cycle. This agrees with previous work that TCE can induce an increase in oxidative DNA damage in rat liver (Toraason et al. 1999). It is reported that rats that received Safrole carcinogen at night, when melatonin level is high, exhibited lower DNA damage than animals treated with the same carcinogen during the day (Tan et al. 1994). Circadian variation in TCE toxicity in rodents under different lighting regimens demonstrated increased liver function parameters and necrosis in the hepatocyte in both 12-h L/12-h D and constant darkness (Motohashi et al. 1990). The present results support these findings and showed remarkable increase in the activity of ALT, AST, and bilirubin content with a severe decrease in albumin concentration in blood, indicating liver toxicity. These changes were confirmed using histopathological evaluation.

Conclusion
The findings from the current study indicate that pineal gland impairment, exposure to continuous light at night, or their combination increase susceptibility to the carcinogenic effects of TCE on the liver. This may be attributable to suppressed antioxidant defenses due to elevated oxidative stress products and inflammatory cytokines in mice.
Author contribution Mohamed A El-Missiry, Mohamed E. Abdraboh, Azza I Othman, Ahmed Nageeb Taha, and Maggie E Amer contributed to the study conception and design. Material preparation, data collection, and analysis were performed, and the first draft of the manuscript was written, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.
Funding Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB). This research received financial support from Competitive Funding Projects, research unit of Mansoura University.
Data availability All data generated or analyzed during this study are included in this published article.

Declarations
Ethics approval and consent to participate The experimental protocol for the treatment of animals under study was carried out following the guidelines approved by the Institutional Animal Ethics Committee (IAEC) of Mansoura University.

Conflict of interest The authors declare no competing interests.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.