Biomarker hepatotoxicity

More and more biomarkers and gene arrays have been identified and getting ready to enter the profiling portfolio to address organotypic toxicity. We refrain from detailed analysis here and refer the reader to the specific chapter addressing hepatotoxicity and hematotoxicity (Chapter 17). 

Drug-induced hepatotoxicity can present in variable manifestations, such as cell death (necrosis, apoptosis), infiammation, degeneration (steatosis), fibrosis/cirrho-sis and the development of tumors. The manifestations of drug toxicity may not be mutually exclusive and may occur sequentially, or in combination. ALT and ALP can be used to generally classify the pattern of liver injury as either hepatocellular (ALT >3x ULN), cholestatic (ALP >2x ULN, ALT/ALP <2) or mixed (elevated ALP and ALT). The successful monitoring of hepatotoxicity would identify cases before irreversible injury occurs. The activity levels of ALT, AST and ALP only increase after hepatic or cholestatic injury has occurred. Waiting for activity levels to exceed the established thresholds may be too late [3]. New biomarkers are needed to monitor/predict the specific sequence of events for different classes of hepatotoxic compounds. 

Changes in serum ALT may not aWays be indicative ofa true hepatotoxic response. Mild dose-related ALT elevations (2x to 3x ULN) are observed in some patients taking lovastatin as a result of an adaptive response [139]. As another example, isoniazid, an anti-tuberculosis agent, leads to a high incidence of ALT and AST elevations, but is tolerated chronically without severe hepatotoxicity. This suggests that more specific and sensitive biomarkers are still needed to predict serious liver injury. 

Apanel of in vivo biomarkers that refiects the different mechanisms of toxicity and that could be used to predict drug response in tissue would be invaluable in hepatotoxicity risk assessment. 

As an example, acetaminophen (APAP) in overdose has been used by several groups to identify hepatotoxicity biomarkers in mice. APAP-induced hepatotoxicity is characterized by hepatic centrilobular necrosis and hepatitis. APAP biotransformation by Phase I enzymes leads to the formation of the reactive metabolite N-acetyl-p-benzoquinone (NAPQI), which can deplete glutathione and form adducts with hepatic proteins (see Section 15.2). Protein adduction primes the hepatocytes for cytokines released by activated macrophages (Kupffer cells) and/or destructive insults by reactive nitrogen species. Although necrosis is recognized as the mode of cell death in APAP overdose, the precise mechanisms are still being elucidated [152]. 

New biomarkers will be useful in hepatotoxicity risk assessment if the data quality and validity can be established. The FDA defines a valid biomarker as one that can be measured in an analytical test system with well-established performance characteristics and has an established scientific framework or body of evidence that elucidates the significance of the test results [160]. Although there is no formerly agreed upon path, biomarker validation should include appropriate end-points for study (i.e., toxicology, histopathology, bioanalytical chemistry, etc.) and dose- and time-dependent measurements. An assessment of species, sex and strain susceptibility is also important to evaluate across species differences. More specific considerations for validation of gene and protein expression technologies are reviewed by Corvi et al. and Rifai et al. [144, 147]. 

Ozer, J.R.M., Shaw, M., Wendy, B. and Schomaker, S. (2009) The current state of serum biomarkers of hepatotoxicity. Toxicology, in press. 

Kikkawa R, Yamamoto T, Fukushima T et al. (2005) Investigation of a hepatotoxicity screening system in primary cell cultures - what biomarkers would need to be addressed to estimate toxicity in conventional and new approaches . J Toxicol Sci 30 61-72... 

The number of useful biomarkers to predict neurotoxicity, hepatotoxicity or cardiovascular toxicity is still rather limited and they are not yet well established as tools in pharmaceutical laboratories. Even though some decent correlations between biomarkers and toxicological events have been demonstrated, " a significant amount of validation work still has to be performed. Eor example, while natriuretic peptides and troponin can be clinical markers for cardiovascular toxicity, preclinical use in... 

Biomarkers of effect would be useful for identifying 1,1-dichloroethane-specific injury (e.g., hepatotoxicity, renal toxicity, neurotoxicity) for short-intermediate-and long-term exposure. 

New biomarker discovery from such studies can lead to early detection of deleterious effects. In a more recent study, researchers validated toxicological protein markers from an in vivo system (rat liver) as well as from an in vitro system (human HepG2 cell line) [86]. They reported a total of 11 protein markers with reactivity toward multiple toxic compounds and no reactivity toward nontoxic compounds. An important conclusion from this work is that cells in culture can be used as an in vitro toxicity testing system to assess hepatotoxicity. However, in the future, a much more extensive study may be required to identify a larger group of toxicology markers to detect more diverse types of toxic reactions. 

Kleno T G, Kiehr B, Baunsgaard D, et al. (2004). Combination of omics data to investigate the mechanism(s) of hydrazine-induced hepatotoxicity in rats and to identify potential biomarkers. Biomarkers. 9 116-138. 

Acetaminophen (paracetamol) is a commonly used analgesic which is hepatotoxic at high doses in humans and in laboratory animals. Toxicity is believed to be mediated by the reactive metabolite N-acetyl-p-benzoquinone imine which binds to protein thiols as 3-(cystein-S-yl)acetaminophen adducts. Ultrasensitive immimoassays for 3-(with parallel elevations in serum adducts and serum levels of the liver-specific transaminase ALT. This suggested that the serum adducts were of hepatic origin and could be monitored as a biomarker of acetaminophen toxicity. Analysis of serum samples from acetaminophen overdose patients demonstrated a positive correlation between immunochemically detectable serum adducts and hepatotoxicity. 

Immunologic techniques were used to study acetaminophen toxicity in mice to elucidate mechanisms of liver toxicity. The hepatotoxicity of acetaminophen is mediated by a reactive metabolite, N-acetyl-p-ben-zoquinone imine (NAPQI). The metabolite binds to protein as 3-(cystein-S-yl)acetaminophen (3-Cys-A) and the amount of binding correlates with toxicity. This covalent binding, and in particular the 3-Cys-A adduct, is the most reliable biomarker of acetaminophen toxicity (9-12). 

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