Search

Blood is an ideal window for viewing our health and disease status. Because blood circulates throughout the entire body and carries secreted, shed, and excreted signature proteins from every organ and tissue type, it is thus possible to use the blood proteome to achieve a comprehensive assessment of multiple-organ physiology and pathology. To date, the blood proteome has been frequently examined for diseases of individual organs; studies on compound insults impacting multiple organs are, however, elusive. We believe that a characterization of peripheral blood for organ-specific proteins affords a powerful strategy to allow early detection, staging, and monitoring of diseases and their treatments at a whole-body level. In this paper we test this hypothesis by examining a mouse model of acetaminophen (APAP)-induced hepatic and extra-hepatic toxicity. We used a glycocapture-assisted global quantitative proteomics (gagQP) approach to study serum proteins and validated our results using Western blot. We discovered in mouse sera both hepatic and extra-hepatic organ-specific proteins. From our validation, it was determined that selected organ-specific proteins had changed their blood concentration during the course of toxicity development and recovery. Interestingly, the peak responding time of proteins specific to different organs varied in a time-course study. The collected molecular information shed light on a complex, dynamic, yet interweaving, multiorgan-enrolled APAP toxicity. The developed technique as well as the identified protein markers is translational to human studies. We hope our work can broaden the utility of blood proteomics in diagnosis and research of the whole-body response to pathogenic cues.

Adverse effects caused by therapeutic drugs are a serious and costly health concern. Despite the body's systemic responses to therapeutics, the liver is often the focus of damage and is usually the focus of studies of toxic effects due to its active roles in the metabolism of xenobiotics. It is extremely difficult, however, to assess systemic responses with currently available methods. Comprehensive cataloging of cell-free circulating RNAs using next-generation sequencing technology may open a window to assess drug-associated adverse effects at the systems level. To explore this potential, we conducted an RNA profiling study using the well-characterized acetaminophen overdose mouse model on liver and plasma with microarray and next-generation sequencing platforms, respectively. After drug treatment, the levels of a number of transcripts, both endogenous and exogenous RNAs, showed significant changes in plasma, reflecting not only the classical liver injury induced by acetaminophen overdose but also damage in tissues other than the liver. The changes in exogenous RNAs also reflect alteration on dieting behavior after acetaminophen overdose. Besides reporting an extensive list of circulating RNA-based biomarker candidates, this study illustrates the possibility of using circulating RNAs to assess global effects of therapeutics. This could also lead to a new approach for a more comprehensive assessment of the efficacy and safety of therapeutics.

Organ-enriched blood proteins, those produced primarily in one organ and secreted or exported to the blood, potentially afford a powerful and specific approach to assessing diseases in their cognate organs. In this paper, we demonstrate that quantification of organ-enriched proteins in the blood offers a new strategy to find biomarkers for diagnosis and assessment of drug-induced liver injury (and presumably the assessment of other liver diseases). We used selected reaction monitoring (SRM) mass spectrometry to quantify 81 liver-enriched proteins plus three aminotransferases (ALT1, AST1, and AST2) in plasma of C57BL/6J and NOD/ShiLtJ mice exposed to acetaminophen or carbon tetrachloride. Plasma concentrations of 49 liver-enriched proteins were perturbed significantly in response to liver injury induced by one or both toxins. We validated four of these toxin-responsive proteins (ALDOB, ASS1, BHMT and GLUD1) by Western blotting. By both assays, these four proteins constitute liver injury markers superior to currently employed markers such as ALT and AST. A similar approach was also successful in human serum where we had analyzed 66 liver-enriched proteins in acetaminophen overdose patients. Of these, 23 proteins were elevated in patients; 15 of 23 overlapped with the concentration-increased proteins in the mouse study. A combination of 5 human proteins, AGXT, ALDOB, CRP, FBP1, and MMP9, provides the best diagnostic performance to distinguish acetaminophen overdose patients from controls (sensitivity: 0.85, specificity: 0.84, accuracy: 85%). These five blood proteins are candidates for detecting acetaminophen-induced liver injury using next-generation diagnostic devices (e.g, microfluidic ELIZA assays).

The hippocampus, a major site of neurogenesis in the adult brain, plays an important role in memory. Based on earlier observations where exposure to high-intensity noise not only caused hearing loss but also impaired memory function, it is conceivably that noise exposure may suppress hippocampal neurogenesis. To evaluate this possibility, nine rats were unilaterally exposed for 2 h to a high-intensity, narrow band of noise centered at 12 kHz at 126 dB SPL. The rats were also screened for noise-induced tinnitus, a potential stressor which may suppress neurogenesis. Five rats developed persistent tinnitus-like behavior while the other four rats showed no signs of tinnitus. Age-matched sham controls showed no signs of hearing loss or tinnitus. The inner ear and hippocampus were evaluated for sensory hair cell loss and neurogenesis 10 weeks post-exposure. All noise exposed rats showed severe loss of sensory hair cells in the noise-exposed ear, but essentially no damage in the unexposed ear. Frontal sections from the hippocampus were immunolabeled for doublecortin to identify neuronal precursor cells, or Ki67 to label proliferating cells. Noise-exposed rats showed a significant reduction of neuronal precursors and fewer dividing cells as compared to sham controls. However, we could not detect any difference between rats with behavioral evidence of tinnitus versus rats without tinnitus. These results show for the first time that high intensity noise exposure not only damages the cochlea but also causes a significant and persistent decrease in hippocampal neurogenesis that may contribute to functional deficits in memory.

The current gold standard for diagnosis of hepatic fibrosis and cirrhosis is the traditional invasive liver biopsy. It is desirable to assess hepatic fibrosis with noninvasive means. Targeted proteomic techniques allow an unbiased assessment of proteins and might be useful to identify proteins related to hepatic fibrosis. We utilized selected reaction monitoring (SRM) targeted proteomics combined with an organ-specific blood protein strategy to identify and quantify 38 liver-specific proteins. A combination of protein C and retinol-binding protein 4 in serum gave promising preliminary results as candidate biomarkers to distinguish patients at different stages of hepatic fibrosis due to chronic infection with hepatitis C virus (HCV). Also, alpha-1-B glycoprotein, complement factor H and insulin-like growth factor binding protein acid labile subunit performed well in distinguishing patients from healthy controls.