Comprehensive Review on Biomarkers in Hepatotoxicity: From Conventional Indicators to Omics‑Driven Discoveries

We aimed to elucidate the frequency of polymorphic genotypes and alleles of patatin-like phospholipase domain containing 3 rs738409 polymorphism and its possible associations with non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis in a cohort from Turkey.We enrolled 200 patients diagnosed with NAFLD and genotyped for rs738409 I148M polymorphism by real-time polymerase chain reaction, particularly by melting curve analysis. SPSS analysis software was used for statistical significance. Continuous variable values were expressed as mean ± standard deviation. Significant statistical level was chosen as p = 0.05.Our results demonstrate in a cohort from Turkey that rs738409 C > G polymorphism (I148M) of patatin-like phospholipase domain containing 3 gene is significantly able to affect individuals to have NAFLD in unadjusted regression model.Consistent with the previous studies in other populations, our study group showed a significantly higher risk of having NAFLD in unadjusted regression model but not in the adjusted model indicating that non-genetic factors such as age and sex may be responsible for the association. However, independent studies need to validate our findings with a larger group of NAFLD patients, as well as in different ethnic cohorts.

A 79-year-old female patient with a secondary pulmonary hypertension received an oral bosentan 62.5 mg twice daily. Her liver function was normal before medication. On the day 57 after begining of the medication, laboratory tests showed aspartate aminotransferase(AST) 44 U/L, alanine aminotransferase (ALT) 43 U/L, gamma glutamyl transferase (γ-GT) 166 U/L, alkaline phosphatase (ALP) 249 U/L, glutamate dehydrogenase(GDH)30.8 U/L, albumin 31.7 g/L, total bilirubin(TBil) 50.0 μmol/L, direct bilirubin(DBil) 23.8 μmol/L , and indirect bilirubin(IBil) 26.2 μmol/L. On the day 62, the patient developed yellowish discoloration of skin and sclera, fatigue and loss of appetite. Laboratory tests showed the following levels: AST 39 U/L, ALT 32 U/L, GGT 276 U/L, ALP 417 U/L, GDH 14.5 U/L, albumin 33.6 g/L, TBil 120.8 μmol/L, DBil 65.3 μmol/L, IBil 55.4 μmol/L. Drug-induced liver injury was diagnosed. Bosentan was withdrawn and hepatoprotective agents were given. Five days after the drug withdrawal, her jaundice disappeared. Thirty-three days after the drug withdrawal, laboratory tests showed AST 25 U/L, ALT 13 U/L, GGT 43 U/L, ALP 125 U/L, GDH 1.8 U/L, albumin 37 g/L, TBil 24.0 μmol/L, DBil 9.2 μmol/L, and IBil 14.8 μmol/L. Key words: Bosentan; Hyperbilirubinemia

Background: Type 2 diabetes mellitus has been linked with abnormal liver function tests. Increased activities of liver enzymes such as aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), ?-glutamyl transferase (GGT) are markers of liver cell injury. Increased activity of these markers is associated with metabolic syndrome, insulin resistance and type 2 diabetes mellitus. This study was aimed to evaluate the significance of liver enzymes such as aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), ?-glutamyl transferase (GGT) in type 2 diabetes mellitus patients. Materials & Methods: Total of 108 cases of type 2 diabetes mellitus patients, comprising of 68 males (62.9%) and 40 females (37%) were included in the study. 110 healthy subjects were includes as controls. Age of the subjects ranged between 27 to 75 years. Serum sample was used for the estimation of study parameters such as FBS, PPBS, AST, ALT, ALP, GGT, Total bilirubin, Direct bilirubin, Total proteins and Albumin. Results: In the present study, mean values of AST, ALT, ALP and GGT were statistically significantly increased in type 2 diabetes mellitus patients when compared to controls (P value 0.001). Conclusion: In this study, we found an association between the increased levels of liver enzymes such as AST, ALT, ALP and GGT in type 2 diabetes mellitus patients. Increased levels of ALT and AST are the surrogate markers for associated non alcoholic fatty liver disease in type 2 diabetes mellitus patients. Hence, testing for AST, ALT, ALP, and GGT should be carried out to screen for underlying non alcoholic fatty liver disease (NAFLD) in type 2 diabetes mellitus patients.

To evaluate hepatobiliary magnetic resonance imaging (MRI) using Gd-EOB-DTPA in relation to various liver function tests in patients with liver disorders. Fifty-one patients with liver disease underwent Gd-EOB-DTPA-enhanced liver MRI. Based on region-of-interest (ROI) analysis, liver signal intensity was calculated using the spleen as reference tissue. Liver-spleen contrast ratio (LSCR) and relative liver enhancement (RLE) were calculated. Serum levels of total bilirubin, gamma glutamyl transpeptidase (GGT), aspartate aminotransferase (AST), alanine aminotransferase (ALT), glutamate dehydrogenase (GLDH), lactate dehydrogenase (LDH), serum albumin level (AL), prothrombin time (PT), creatinine (CR) as well as international normalised ratio (INR) and model for end-stage liver disease (MELD) score were tested for correlation with LSCR and RLE. Pre-contrast LSCR values correlated with total bilirubin (r = -0.39; p = 0.005), GGT (r = -0.37; p = 0.009), AST (r = -0.38; p = 0.013), ALT (r = -0.29; p = 0.046), PT (r = 0.52; p < 0.001), GLDH (r = -0.55; p = 0.044), INR (r = -0.42; p = 0.003), and MELD Score (r = -0.53; p < 0.001). After administration of Gd-EOB-DTPA bilirubin (r = -0.45; p = 0.001), GGT (r = -0.40; p = 0.004), PT (r = 0.54; p < 0.001), AST (r = -0.46; p = 0.002), ALT (r = -0.31; p = 0.030), INR (r = -0.45; p = 0.001) and MELD Score (r = -0.56; p < 0.001) significantly correlated with LSCR. RLE correlated with bilirubin (r = -0.40; p = 0.004), AST (r = -0.38; p = 0.013), PT (r = 0.42; p = 0.003), GGT (r = -0.33; p = 0.020), INR (r = -0.36; p = 0.011) and MELD Score (r = -0.43; p = 0.003). Liver-spleen contrast ratio and relative liver enhancement using Gd-EOB-DTPA correlate with a number of routinely used biochemical liver function tests, suggesting that hepatobiliary MRI may serve as a valuable biomarker for liver function. The strongest correlation with liver enhancement was found for the MELD Score. • Relative enhancement (RLE) of Gd-EOB-DTPA is related to biochemical liver function tests. • Correlation of RLE with bilirubin, ALT, AST, GGT, INR and MELD Score is reverse. • The correlation of relative liver enhancement with prothrombin time is positive. • AST, ALT, GLDH, prothrombin time, INR and MELD Score correlate with pre-contrast liver-spleen contrast ratio. • Such biomarkers may help to evaluate liver function.

Effects of Propofol on the Outcomes of Rats with Sepsis

To investigate the role of high mobility group box 1 (HMGB1) in hepatic endoplasmic reticulum stress (ERS) in rats with trauma. Sixty SPF Sprague-Dawley (SD) rats were randomly divided into groups (n = 6). The rat model of liver injury following traumatic stress was established by continuous compressing the bilateral hind-limbs of rats for 3 hours and then intermittent compressing and decompressing for 30 minutes respectively three times with standard weight of 15 kg. The experiment 1 was divided into two groups: control group and 6, 18, 30 hours after crush. The experiment 2 was divided into control group, crush model group (18 hours after crush), HMGB1 inhibitor sodium butyrate (SB) or ethyl pyruvate (EP) groups, and SB or EP treatment groups (500 mg/kg SB solution or 40 mg/kg EP solution was injected intraperitoneally after 3 hours crush). The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum were measured with automatic biochemistry analyzer. Histopathological severity of liver injury was assessed by hematoxylin and eosin (HE) staining. The expressions of HMGB1 and ERS-related proteins were detected with Western Blot. The expression and translocation of HMGB1 in liver tissue were evaluated by immuno-histochemical technique. (1) Compared with the control group, the pathological changes of liver injury, the levels of AST and ALT in serum and protein expression of HMGB1 as well as ERS-related proteins such as glucose regulated protein 78 (GRP78), caspase-12, and inositol-requiring enzyme 1α (IRE1α) in liver tissue were significantly increased after traumatic stress, and reached the peak at 18 hours. The expression of C/EBP-homologous protein (CHOP) was increased in a time-dependent manner and peaked at 30 hours after crush. Immunohistochemistry showed that HMGB1 expression increased at 6 hours after crush, some HMGB1 shifted from nucleus to cytoplasm, and the expression was more obvious at 18 hours. (2) Compared with crush model group, the expressions of HMGB1 and ERS-related proteins were significantly decreased following the administration of HMGB1 inhibitors SB or EP (HMGB1/β-actin: 0.703±0.213, 0.512±0.075 vs. 1.041±0.186; GRP78/β-actin: 0.614±0.052, 0.450±0.115 vs. 0.847±0.120; caspase-12/β-actin: 0.636±0.066, 0.812±0.142 vs. 1.086±0.130; CHOP/β-actin: 0.314±0.046, 0.621±0.123 vs. 0.996±0.764; IRE1α/β-actin: 0.473±0.033, 0.519±0.094 vs. 0.742±0.054, all P < 0.05), the levels of serum AST and ALT were significantly decreased [AST (U/L): 1 030.50±427.73, 1 414.50±347.86 vs. 2 122.20±322.76; ALT (U/L): 285.75±11.30, 368.50±80.58 vs. 473.80±33.54, all P < 0.01], the degree of acute liver injury was reduced. Only SB or EP could not affect the parameters mentioned above. HMGB1-ERS pathway was involved in mediating traumatic stress-induced acute liver injury in rats.

Heatstroke is generally considered as a sepsis-like syndrome induced by hyperthermia leading to multiorgan dysfunction. High-mobility group box 1 (HMGB1) has recently been identified as a mediator of systemic inflammation leading to multiorgan dysfunction in sepsis and nonsepsis. Elevation of plasma HMGB1 in heatstroke has been suggested in experimental models and clinical patients. By far, whether HMGB1 could be a potential therapeutic target in heatstroke is unknown. The objectives of this study are to use HMGB1 monoclonal antibody to specifically inhibit the activity of extracellular HMGB1 and to observe the possible protection of liver injury in a rat heatstroke model. After treatment with neutralizing antibodies to HMGB1, rats were exposed to a high-temperature and high-humidity environment. At the time of heatstroke onset, the plasma and liver cytoplasm HMGB1 levels were detected by enzyme-linked immunosorbent assay. The histopathology of liver tissue was observed under light microscopy and transmission electron microscopy. Plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were determined using the commercially available kits. Plasma tumor necrosis factor-α, interleukin-1β (IL-1β), and IL-6 were determined using enzyme-linked immunosorbent assay kits. HMGB1 levels in plasma and liver cytoplasm were both elevated in heatstroke rats, which were both associated with increased plasma ALT and AST levels. Histopathologic results showed that HMGB1 monoclonal antibody pretreatment could obviously alleviate the pathologic impairments of heatstroke rats. HMGB1 monoclonal antibody pretreatment could also downregulate plasma AST and ALT levels in heatstroke rats. Plasma tumor necrosis factor-α, IL-1β, and IL-6 levels in heatstroke rats were elevated, which could be significantly suppressed by HMGB1 antibody pretreatment. HMGB1 could be a potentially effective treatment target in heatstroke. The pathogenic mechanism of heatstoke is complicated, which needs comprehensive prevention and treatment.

Background Drug-induced hepatotoxicity represents a major clinical problem worldwide. Hepatotoxicity may be caused by drugs, chemicals used in laboratories and industries or natural products and rodenticides such as metal phosphides. Early identification of hepatotoxicity would facilitate patient- individualized treatment strategies. New biomarkers (HMGB1and K18) used to identify subsequent acute liver injury (ALI) in patients on admission to the hospital. Objectives This study aims to assess the role of keratin 18 and high mobility group box 1 biomarkers in early prediction of drugs and chemicals induced hepatotoxicity for better outcome and to compare these new liver biomarkers with the currently used parameters (ALT &amp; AST) in the early prediction of drugs and chemicals induced hepatotoxicity. Methods This prospective study was conducted on 50 adult patients of both sex presented to the PCC- ASUH with acute intoxication of hepatotoxic drugs or chemicals. For all patients, we measured high mobility group box-1 (HMGB1), keratin-18 (K18), AST and ALT on admission then patients were followed- up for development of liver injury . Subjects were classified into group I (control group), group II (no liver affection) and group III (with liver affection). Receiver operator characteristic (ROC) curve was used to compare sensitivity and specificity to report liver injury versus alanine transaminase (ALT) and aspartate transaminase (AST). Results This study showed that HMGB1 and K18 were much more elevated in patients who developed liver injury in the first hospital presentation when ALT and AST were within normal ranges with a highly significant difference between the hepatotoxic and non-hepatotoxic group. ROC analysis showed that HMGB1 and K18 were more sensitive than ALT and AST and more specific than AST in predicting ALI. Conclusion Elevations in plasma HMGB1, and K18 identified subsequent ALI development in patients on admission to the hospital, soon after drug and chemical overdose, and in patients with ALT and AST in the normal range.

Patterns and prediction of liver injury with persistent cholestasis in survivors of severe SARS-CoV-2 infection

Observing the dynamic change characteristics of serum liver function indexes in occupational dermatitis medicamentosa-like of trichloroethylene patients with liver damage, we can underlie for guiding therapy, prognosis and mechanism of dermatitis medicamentosa-like of trichloroethylene patients with liver damage. We collected serum of 10 cases of occupational dermatitis medicamentosa-like of trichloro-ethylene patients with liver damage from different time points since they were hospitalized, using automatic biochemistry analyzer to detect total protein (TP), albumin (ALB), total bilirubin (TBIL), direct bilirubin (DBIL), indirect bilirubin (IBIL), alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transpeptidase (GGT), alkaline phosphatase (ALP), albumin/globulin ratio etc 11 liver function biochemical indicators. We used Excel to establish database, professional drawing software gnuplot to draw dynamic variation diagram of each index. The variation range of 11 liver function indexes of 10 cases was TP 43.2-74.2 g/L, ALB 24.6-44.6 g/L, A/G 0.77-2.10, TBIL 3.7-268.2 umol/L, DBIL 1.0-166.0 umol/L, IBIL 2.4 -167.5 umol/L, ALT 11-5985 U/L, AST 14-5586 U/L, GGT 15-1500 U/L, ALP 35-309 U/L, S/L 0.07-1.94, respectively. TBIL, DBIL, ALT, AST, GGT, ALP concentration significantly increased, especially ALT, AST, GGT, ALT topped 5985 U/L, AST topped 5586 U/L, GGT topped 1500 U/L. But TP, ALB and S/L significantly decreased, TP lowest to 43.2 g/L, S/L lowest to 0.07. A/G basically remained unchanged, but IBIL didn't change regularly. The early liver damage in dermatitis medicamentosa-like of trichloroethylene patients was serious, and repeatedly attacked, so we should lead to enough attention to the clinical work and prevention. This also provided the basis for studying the mechanism of trichloroethylene poisoning.

This retrospective study evaluated the sensitivity and clinical importance of liver parameters (alanine aminotransferase [ALT], alkaline phosphatase [AP], aspartate aminotransferase [AST], glutamate dehydrogenase [GLDH], γ-glutamyltransferase [GGT], glucose, albumin, total protein, bilirubin, and urea) for diagnosing hepatopathies (hepatic lipidosis, inflammation, diseases of the bile duct, neoplasia, cirrhosis, fibrosis, other liver diseases) in rabbits. The laboratory results of 77 rabbits with hepatopathies diagnosed via cytological or histopathological examination were investigated by assessing frequency distributions, associations between liver parameters and different hepatopathies, and intercorrelations between parameters. The most frequent liver disease was hepatic lipidosis (40/77), followed by inflammation (3/77). Aspartate aminotransferase was the parameter most commonly increased (n = 20/77, 70.0% above the reference interval), whereas AP activity never exceeded the reference interval. Significant (p 0.9) were found between AST/ALT, GGT/ALT, GGT/AST, and bilirubin/GGT, and significant but lower correlations (p ≤ 0.001, r = 0.–0.9) were detected for GLDH/ALT, and GLDH/AST. The study showed that AST, GLDH, ALT, and GGT, in contrast to AP, represent suitable parameters for detecting hepatopathies in rabbits.

BACKGROUNDCyclophosphamide (CP) is a potent chemotherapeutic and immunosuppressant agent, but its hepatotoxicity remains a significant concern. Ambroxol (ABX) is a mucolytic agent with emerging beneficial effects against oxidative stress and inflammation.AIMTo investigate the hepatoprotective effects of ABX against CP-induced liver injury, focusing on oxidative stress, inflammation, and the possible role of cytoglobin, thioredoxin reductase 1 (TXNRD1) and high-mobility group box 1 (HMGB1).METHODSABX (20 mg/kg) was orally administered for 7 days, and the rats received a single injection of CP (100 mg/kg) on day 5. Blood and liver samples were collected for analyses, and the affinity of ABX towards cytoglobin, TXNRD1, and HMGB1 was evaluated using molecular docking.RESULTSCP administration significantly elevated alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase, reduced albumin, and caused multiple histopathological alterations in the liver. ABX effectively restored liver function biomarkers and attenuated histopathological alterations. CP-induced oxidative stress was evidenced by increased malondialdehyde and decreased glutathione and antioxidant enzyme activities, all of which were ameliorated by ABX. CP upregulated toll-like receptor 4 (TLR-4), nuclear factor-kappaB (NF-κB) p65 and pro-inflammatory cytokines, while downregulating cytoglobin, TXNRD1 and HMGB1. ABX suppressed TLR-4/NF-κB signaling and pro-inflammatory cytokines, and upregulated cytoglobin, TXNRD1 and HMGB1. In silico molecular docking revealed the affinity of ABX to bind with cytoglobin, TXNRD1, and HMGB1.CONCLUSIONABX protects against CP hepatotoxicity by mitigating oxidative stress, suppressing TLR-4/NF-κB signaling, and upregulating cytoglobin, TXNRD1 and HMGB1. ABX showed binding affinity towards cytoglobin, TXNRD1 and HMGB1. These findings suggest that ABX has therapeutic potential in alleviating hepatotoxicity associated with CP treatment.

Serum enzyme tests in hepatosplenic schistosomiasis

We previously reported that high mobility group box 1 (HMGB1), a danger-associated molecular pattern (DAMP), increases intracellular iron levels in the postischemic brain by upregulating hepcidin, a key regulator of iron homeostasis, triggering ferroptosis. Since hepatocytes are the primary cells that produce hepcidin and control systemic iron levels, we investigated whether cerebral ischemia induces hepcidin upregulation in hepatocytes. Following middle cerebral artery occlusion (MCAO) in a rodent model, significant liver injury was observed. This injury was evidenced by significantly elevated Eckhoff’s scores and increased serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Additionally, total iron levels were significantly elevated in the liver, with intracellular iron accumulation detected in hepatocytes. Hepcidin expression in the liver, which is primarily localized in hepatocytes, increased significantly starting at 3 h after MCAO and continued to increase rapidly, reaching a peak at 24 h. Interestingly, HMGB1 levels in the liver were also significantly elevated after MCAO, with the disulfide form of HMGB1 being the major subtype. In vitro experiments using AML12 hepatocytes showed that recombinant disulfide HMGB1 significantly upregulated hepcidin expression in a Toll-like receptor 4 (TLR4)- and RAGE-dependent manner. Furthermore, treatment with a ROS scavenger and a peptide HMGB1 antagonist revealed that both ROS generation and HMGB1 induction contributed to hepatocyte activation and liver damage following MCAO–reperfusion. In conclusion, this study revealed that cerebral ischemia triggers hepatocyte activation and liver injury. HMGB1 potently induces hepcidin not only in the brain but also in the liver, thereby influencing systemic iron homeostasis following ischemic stroke.

Background/Aims: Interferon regulatory factor 1(IRF-1) and high mobility group box 1(HMGB1) have been independently identified as being key players in hepatic ischemia-reperfusion injury (IRI). We attempted to determine whether IRF-1 activates autophagy to aggravate hepatic IRI by increasing HMGB1 release. Methods: The hepatic IRI model was generated in C57BL/6 mice, euthanized at 2, 6, 12 or 24 h after reperfusion. To examine the effects of HMGB1 release inhibition, Glycyrrhiza acid (GA) was administered to the mice and at six hours after injectiont. AML12 cells were immersed in mineral oil for 90 min and then cultured in complete Dulbecco’s Modified Eagle’s Medium (DMEM)/F12 to simulate IRI. AML12 cells were treated with IRF-1 siRNA, Ad-IRF-1 or GA. The serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT), as well as histological changes were examined. Next, autophagic vacuoles were detected by transmission electron microscopy (TEM) or LC3 dots. The expression of IRF-1 and HMGB1 mRNA were measured by real-time polymerase chain reaction. The expression of IRF-1, microtubule-associated protein 1 light chain 3 (LC3), Bcl-2, Beclin 1, HMGB1 were detected by western blotting or immunohistochemistry. Results: The levels of hepatic IRF-1, mRNA and protein were significantly increased in livers after exposure to IRI, together with, IRI-induced increase of HMGB1 mRNA and release of HMGB1 in liver tissue. Knockout of IRF-1 decreased expression and release of HMGB1 in liver, and inhibiting the release of HMGB1 could alleviate hepatic IRI. In addition, knockout of IRF-1 downregulated LC3II and Beclin1, while number of autophagosomes or LC3 dots were increased. Up-regulating IRF-1 expression could increase the levels of LC3Ⅱ expression in AML12 cells after exposure to IRI. The levels of HMGB1 in Ad-IRF-1 transfected AML12 cell supernatants increased, together with number of LC3 dots increasing. However, GA could inhibit both Ad-IRF-1 induced HMGB1 release and the increase in the number of LC3 dots. Conclusions: IRF-1 activates autophagy to aggravate hepatic IRI by increasing HMGB1 release.