An Overview on Biological Markers in Reproductive and Developmental Toxicology: Concepts, Definitions and Use in Risk Assessment

Reproduction and development are complex couple-dependent processes. Risk assessment for these health outcomes requires the use of biomarkers to link exposures to disease. Biological markers of susceptability, external dose, internal dose, biologically effective dose, early or late biological responses, altered reproductive or developmental function, and reproductive or developmental disease are introduced. Using these biomarkers it is possible to define a biologically based risk assessment methodology for reproductive and developmental toxicity. Risk assessment for reproductive toxicity requires definition of male and female fecundity, couple-specific factors, spontaneous abortion, rate, and other factors. Using using sperm count as a biomarker for male fecundity, an example of a reproductive risk assessment using biomarkers is performed.

Biological Markers on Human Pregnancy

This article reviews a selected set of recently described pregnancy-associated proteins which possess potential for both signaling pregnancy onset and monitoring its course. These molecules are compared and contrasted with human chorionic gonadotropin, the first pregnancy-associated protein to be discovered, and the standard biomarker of pregnancy to which all others must still be referenced. Recent advances in hCG research have focused on the structural determination and diagnostic significance of the subunits and fragments of the hCG molecule, particularly in urine. An outline of the potential utility of this approach is also presented.

Metabolomic Analysis in Saliva and Different Brain Regions of Older Mice with Postoperative Delirium Behaviors

Objective Postoperative delirium(POD)has become a critical challenge with severe consequences and increased incidences as the global population ages.However,the underlying mechanism is yet unknown.Our study aimed to explore the changes in metabolites in three specific brain regions and saliva of older mice with postoperative delirium behavior and to identify potential non-invasive biomarkers.Methods Eighteen-month-old male C57/BL6 mice were randomly assigned to the anesthesia/surgery or control group.Behavioral tests were conducted 24 h before surgery and 6,9,and 24 h after surgery.Complement C3(C3)and S100 calcium-binding protein B protein(S100beta)levels were measured in the hippocampus,and a metabolomics analysis was performed on saliva,hippocampus,cortex,and amygdala samples.Results In total,43,33,38,and 14 differential metabolites were detected in the saliva,hippocampus,cortex,and amygdala,respectively.“Pyruvate”“alpha-linolenic acid”and“2-oleoyl-1-palmitoy-snglycero-3-phosphocholine”are enriched in one common pathway and may be potential non-invasive biomarkers for POD.Common changes were observed in the three brain regions,with the upregulation of 1-methylhistidine and downregulation of D-glutamine.Conclusion Dysfunctions in energy metabolism,oxidative stress,and neurotransmitter dysregulation are implicated in the development of POD.The identification of changes in the level of salivary metabolite biomarkers could aid in the development of noninvasive diagnostic methods for POD.

Cell-free plasma hypermethylated CASZ1, CDH13 and ING2 are promising biomarkers of esophageal cancer

Identifying sensitive and specific biomarkers for early detection of cancer is immensely imperative for early diagnosis and treatment and better clinical outcome of cancer patients. This study aimed to construct a specific DNA methylation pattern of cancer suppressor genes and explore the feasibility of applying cell-free DNA based methylation as a biomarker for early diagnosis of esophageal squamous cell carcinoma(ESCC). We recruited early stage ESCC patients from Yangzhong County, China. The Illumina Infinium 450 K Methylation BeadChip was used to construct a genome-wide DNA methylation profile. Then, differentiated genes were selected for the validation study using the Sequenom MassARRAY platform. The frequency of methylation was compared between cancer tissues, matched cell-free DNAs and normal controls. The specific methylation profiles were constructed, and the sensitivity and specificity were calculated. Seven CG sites in three genes CASZ1, CDH13 and ING2 were significantly hypermethylated in ESCC as compared with normal controls. A significant correlation was found between the methylation of DNA extracted from cancer tissues and matched plasma cell-free DNA, either for individual CG site or for cumulative methylation analysis. The sensitivity and specificity reached 100% at an appropriate cut-point using these specific methylation biomarkers. This study revealed that aberrant DNA methylation is a promising biomarker for molecular diagnosis of esophageal cancer. Hypermethylation of CASZ1,CDH13 and ING2 detected in plasma cell-free DNA can be applied as a potential noninvasive biomarker for diagnosis of esophageal cancer.

Monocyte chemoattractant protein-1, transforming growth factor-β1, nerve growth factor, resistin and hyaluronic acid as serum markers：comparison between recurrent acute and chronic pancreatitis

BACKGROUND： Diagnostic parameters that can predict the presence of chronic pancreatitis（CP） in patients with recurrent pain due to pancreatitis would help to direct appropriate therapy. This study aimed to compare the serum levels of monocyte chemoattractant protein-1（MCP-1）, transforming growth factor-β1（TGF-β1）, nerve growth factor（NGF）, resistin and hyaluronic acid（HA） in patients with recurrent acute pancreatitis（RAP） and CP to assess their ability to differentiate the two conditions.METHODS： Levels of serum markers assessed by enzymelinked immunosorbent assay（ELISA） were prospectively compared in consecutive patients with RAP, CP and in controls and stepwise discriminant analysis was performed to identify the markers differentiating RAP from CP.RESULTS： One hundred and thirteen consecutive patients（RAP=32, CP=81） and 78 healthy controls were prospectively enrolled. The mean（SD） age of the patients was 32.0（14.0）years; 89（78.8%） were male. All markers were significantly higher in CP patients than in the controls（P〈0.001）; MCP-1NGF and HA were significantly higher in RAP patients than in the controls（P〈0.001）. Stepwise discriminant analysis showed significant difference（P=0.002） between RAP and CP for resistin with an accuracy of 61.9%, discriminant scores of ≤-0.479 and ≥0.189 indicating RAP and CP, respectively. The other markers had no differential value between RAP and CP.CONCLUSION： Serum resistin is a promising marker to differentiate between RAP and CP and needs validation in future studies, especially in those with early CP.

Evolution of Biochemical, Hematological, Inflammatory and Immunological Markers among Person Living with HIV-1 on ART in Burkina Faso

In Human immunodeficiency virus infected (HIV+) patients, deregulation of regulatory T (Treg) cells can be deleterious for the development of an efficient anti-HIV specific immune response with an inappropriate immune activation despite anti-retroviral therapy (ART). In addition, infection and ART have the potential to cause hematological and biochemical abnormalities that can lead to the discontinuation of ART. The aim of this study was to assess hematological, biochemical and immunological abnormalities in HIV+ patients’ naïve to ART and 6 months after ART initiation. In a cross-sectional study, 11 HIV+ patients and 09 healthy individuals (control group) were voluntarily recruited. At inclusion, blood samples were taken before administration of ART. All hematological, biochemical, immunological parameters and viral load were measured and assessed at inclusion (M0) and three and/or six months later (M3, M6). Higher level of white blood cells and CD4+ T lymphocytes (p = 0.032, 0.038 respectively) were observed in HIV+ patients. ART also had significantly effect on the level of red blood cells (p = 0.04) and Hb (p = 0.015). The inflammatory markers, erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) that were significantly increased by HIV infection, tended to decrease for ESR and revert to levels similar to those of control for CRP. Analysis of CD4+CD25+ cells and CD4+Foxp3+CD25+ showed significantly elevated levels of activated form of all CD4+ cells and Treg cells. Interestingly, six months after initiation of ART, the average percentage of CD4+CD25+ was not significantly different from control group (p = 0.382). Our study provides information about the evolution of the activated form of all CD4 cells and Treg cells and points out the necessity to monitor hematological and biochemical parameters in order to detect and prevent toxicity, improve the quality of life and reduce the risk of mortality.

Endotoxins in the prostatic secretions of chronic prostatitis patients:a need for further biomarkers through the use of proteomics

Dear Sir,Dai et al.[1] must be commended on their useful investigation of the clinical significance of endotoxins in the expressed prostatic secretions （EPS） of chronic prostatitis （CP） patients. However, we take issue with their conclusion. The conclusion that EPS endotoxin determination is helpful in diagnostic confirmation is plausible,

The diagnostic value of multiple tumor markers in malignantovarian neoplasms

Objective:To study the diagnostic value of multiple tumor markers in malignant ovarian neoplasm.Methods:Sera obtained from 430 patients with ovarian masses (110 cases were malignant ovarian tumors,320 cases were benign ovarian tumors) before operation,and from 50 healthy women as control.Serologic examination of tumor markers included CA125,TSGF,SA,CEA,AFP,HCG and Fer.Results:The serum levels of CA125,TSGF,SA and Fer in patients with ovarian cancer were higher than those in patients with benign ovarian tumors (P<0.05),also in control group (P<0.05).In the diagnostic value of application for malignant ovarian neoplasm,CA125,TSGF and SA were better than the others.The sensitivity,specificity and accuracy in diagnosis of ovarian cancer were 86.4%,82.8%and 83.7% respectively for CA125 alone,78.2%,81.3%and 80.5% for TSGF alone,74.5%,81.9%and 80.0% for SA alone,whereas 95.5%,45.6%and 58.4% for multiple tumor markers combined in which 1 or more indices showed positive,93.6%,80.6%and 84.0% for that in which 2 or more indices showed positive,and 87.3%,90.3%and 89.5% for that in which 3 or more indices show positive.Conclusion:multiple tumor markers examination could improve the diagnosis of ovarian cancer,and examination of CA125,TSGF and SA combined is most ideal.

Commentary on: Mobley AJ, Lam YW, Lau KM, Pais VM, Lesperance JO, Steadman B, et al. Monitoring the serological Proteome Colon, the latest modality in prostate cancer detection. J Urol 2004; 172: 331-7.

This manuscript examines the utility, utilizing the Ciphergen Protein Biosystem II, to develop a fingerprint for the diagnosis of prostate cancer. The investigators compared samples from control individuals as well as those with prostate cancer. In doing so, they utilize several chip platforms on which to examine the resulting

Present and future possibilities for early diagnosis of hepatocellular carcinoma

Hepatocellular carcinoma(HCC) represents the fifth most common cancer in the world,and the third most frequent oncological cause of death.The incidence of HCC is on the increase.HCC typically develops in patients with chronic liver diseases,and cirrhosis,usually with viral etiology,is the strongest predisposing factor.Nowadays HCC diagnosis is a multistage process including clinical,laboratory,imaging and pathological examinations.The prognosis of HCC is mostly poor,because of detection at an advanced,non-resectable stage.Potentially curative treatment(surgery) is limited and really possible only for cases with small HCC malignancies.For this reason,more effective surveillance strategies should be used to screen for early occurrence of HCC targeted to the population at risk.So far,the generally accepted serological marker is α-fetoprotein(AFP).Its diagnostic accuracy is unsatisfactory and questionable because of low sensitivity,therefore there is a strong demand by clinicians for new HCC-specific biomarkers.In this review,we will focus on other biomarkers that seem to improve HCC diagnosis,such as AFP-L3,des-γ-carboxyprothrombin,α-l-fucosidase,,γ-glutamyl transferase,glypican-3,squamous cell carcinoma antigen,a new generation of immunoglobulin M-immunocomplexes,and very promising geneexpression profiling.

Neutral Red Retention by Earthworm Coelomocytes: A Biomarker of Cadmium Contamination in Soil

The earthworm Metaphire posthuma were used as a model to assess the toxic potential of cadmium incorporated into the soil by environmental or human activities. The retention period of neutral red in the lysosomes of the coelomocytes was used as a biomarker. The viability of harvested coelomocytes by a non_invasive extrusion protocol was 93% with no alteration by the dye during experimentation. The control cells retained dye for 119 and 121 min in normal soil and KCl, respectively, whereas a linear decline in the retention time in the treated earthworm coelomocytes was observed. This illustrated that the presence of cadmium caused damage to the lysosomes of the coelomocytes.

Increase of beta-amyloid and C-reactive protein in liver transplant recipients with postoperative cognitive dysfunction

BACKGROUND: Postoperative cognitive dysfunction (POCD) is an adverse condition characterized by declined cognitive functions following surgeries and anesthesia. POCD has been associated with increased hospital stay and mortality. There are histological similarities to Alzheimer’s disease. Most early studies were conducted in patients receiving cardiac surgery. Since there is no information about POCD in liver transplant recipients, we measured the incidence of POCD in patients after liver transplantation and examined the correlation between neurological dysfunction and biological markers of dementia- based diseases. METHODS: We studied 25 patients who had a liver transplan- tation between July 2008 and February 2009. Patients with prior encephalopathy or risk factors associated with the development of POCD were excluded from the study. Five validated neuropsychiatric tests were used for diagnosis. The diagnosis was based on one standard deviation decline in two of the five neuropsychiatric tests. The correlation between patient variables and the development of POCD was examined. Serum levels of beta-amyloid and C-reactive protein were measured by standard ELISA and compared between patients with and without POCD. RESULTS: POCD was present in 11 (44%) of the 25 patients. Patients with POCD had significantly higher MELD scores, were more often Child-Pugh class C and received more blood transfusion during surgery. The serum beta-amyloid protein and C-reactive protein concentrations were significantly increased at 24 hours after surgery in the POCD group.CONCLUSIONS: The incidence of POCD in our group of liver transplant patients was greater than that reported in other surgical patients. The increase in the serum biomarkers of dementia in the POCD patients supports the hypothesis that chronic cognitive defects are due to a process similar to that seen in Alzheimer’s disease.

Circulating tumor cell isolation:the assets of filtration methods with polycarbonate track-etched filters

Circulating tumor cells （CTCs） arise from primary or secondary tumors and enter the bloodstream by active or passive intravasation. Given the low number of CTCs, enrichment is necessary for detection. Filtration methods are based on selection of CTCs by size using a filter with 6.5 to 8 pm pores. After coloration, collected CTCs are evaluated according to morphological criteria. Immunophenotyping and fluorescence in situ hybridization techniques may be used. Selected CTCs can also be cultivated in vitro to provide more material. Analysis of genomic mutations is difficult because it requires adapted techniques due to limited DNA materials. Filtration-selected CTCs have shown prognostic value in many studies but multicentric validating trials are mandatory to strengthen this assessment. Other clinical applications are promising such as follow-up, therapy response prediction and diagnosis. Microfluidic emerging systems could optimize filtration-selected CTCs by increasing selection accuracy.

Advancement in biochemical assays in andrology

Determination of markers of sperm function, accessory sex gland secretion and silent male genital tractinflammation is of considerable diagnostic value in the evaluation of male infertility. The introduction of biochemicaltests into the analysis of male factor has the advantage that standardized assays with a coefficient of variationcharacteristic of clinical chemistry are performed, in contrast to biological test systems with a large variability.Biochemical parameters may be used in clinical practice to evaluate the sperm fertilizing capacity (acrosin, aniline blue,ROS), to characterize male accessory sex gland secretions (fructose, α-glucosidase, PSA), and to identify men withsilent genital tract inflammation (elastase, C'3 complement component, coeruloplasmin, IgA, IgG, ROS).

Clinical significance of EGF and EGFR expression changes in cryptorchid boys

AIM: To explore the changes of epidermal growth factor (EGF) and epidermal growth factor receptor (EGFR) expressions in cryptorchid children and their clinical significance. METHODS: The level of serum EGF was measured by radioimmunoassay (RIA) and the expression of EGFR by immunohistochemistry. RESULTS: (1) The level of serum EGF was significantly lower in cryptorchid children than in normal subjects at age group of 5-9 years (P

The prognostic value of preoperative serum levels of CEA,CA19-9 and CA72-4 in patients with colorectal cancer

INTRODUCTIONCarcinoembryonic antigen ( CEA) , originally described by Gold and Freedman [1] in 1965, is now an acknowledged member of immunoglobulin superfamily[2],with a role as an intracellular adhesion molecule[3].Carbohydrate antigen 19-9(CA19-9), obtained with a monoclonal antibody produced by immunizing a monoclonal antibody produced by immunizing a mouse with a colonic cancer cell line in 1979[4],is a ligand for E-selectin that plays an important role in the addhesion of cancer cells to endothelial cells [5,6].

CT Texture Analysis: A Potential Biomarker for Evaluating KRAS Mutational Status in Colorectal Cancer

Objective Texture analysis is deemed to reflect intratumor heterogeneity invisible to the naked eyes.The aim of this study was to evaluate the feasibility of assessing the KRAS mutational status in colorectal cancer(CRC)patients using CT texture analysis.Methods This retrospective study included 92 patients who had histopathologically confirmed CRC and underwent preoperative contrast-enhanced CT examinations.The patients were assigned into a training cohort(n=51)and a validation cohort(n=41).We placed the region of interest in the tumour regions on the selected axial images using software of lexRad to extract a series of quantitative parameters based on the spatial scaling factors(SSFs),including mean,standard deviation(SD),entropy,mean of positive pixels(MPP),skewness,and kurtosis.The texture parameters and clinical characteristics(age,gender,tumour location,histopathology;tumour size,1 N,M stages)were compared between the mutated and wild-type KRAS patient groups in training cohort and validation cohort.Before building the multiple feature classifier,we calculated the correlations of the features using Pearsons correlation coefficient,and if any two features were significantly correlated,the one with lower AUC was removed.Ultimately,only the most discriminative isolated features were combined to train a supporting vector machine(SVM)classifier.The receiver operating characteristic(ROC)curve was processed for evaluating the diagnostic efficiency of texture parameters in differentiating CRC patients with mutated KRAS from those with wild-type KRAS.Results None of the clinical characteristics were significant different between CRC patients with wild-type KRAS and mutated KRAS in both cohorts.For predicting the expression of mutated KRAS in CRC patients,the perfect model which combined skewness on SSF 5 by unenhanced CT,entropy on SSF 2,skewness and kurtosis on SSF 0,and kurtosis and mean on SSF 3 by enhanced CT,showed a desirable AUC of 0.951(95%CI:0.895-1,P<0.001),with a sensitivity of 88.9%and a specificity of 91.7%,when the cut-off value was 0.46 in the training cohort;while in the validation cohort,the AUC value was 0.995(95%CI:0.982-1,P<0.001),the sensitivity was 100%,and the specificity was 93.7%when the cut-off value was 0.28.Conclusion It is feasible to evaluate the KRAS mutational status in CRC using CT texture analysis.

Using Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to analyze differentially expressed brain polypeptides in scrapie strain 22L-infected BALB/c mice

Differentialiy expressed polypeptides in the brain of a BALB/c mouse model infected with scrapie strain 22L were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The results showed that 21 peptides were down-regulated, with peptides of mass-to-charge ratio 758.772 5 and mass-to-charge ratio 5 432.206 9, demonstrating the most significant decreases. These finding suggest that these peptides are candidate biomarkers and may play an important role in the pathogenesis of prion disease.

Serum Protein Fingerprint of Patients with Pancreatic Cancer by SELDI Technology

Objective： To study the serum protein fingerprint of patients with pancreatic cancer and to screen for protein molecules closely related to pancreatic cancer during the onset and progression of the disease using surface-enhanced laser desorption and ionization time of fight mass spectrometry （SELDI-TOF-MS）. Methods： Serum samples from 20 pancreatic cancers, 20 healthy volunteers and 18 patients with other pancreatic diseases. WCX magnetic beans and PBSII-C protein chips reader （Ciphergen Biosystems Ins.） were used. The protein fingerprint expression of all the Serum samples and the resulting profiles between cancer and normal were analyzed with BiomarkerWizard system. Results： Agroup ofproteomic peaks were detected. Four differently expressed potential biomarkers were identified with the relative molecular weights of 5705 Da, 4935 Da, 5318 Da and 3243 Da. Among them, two proteins with m/z5705, 5318Da down-regulated, and two proteins with m/z 4935, 3243 Da were up-regulated in pancreatic cancers. Conclusion： SELDI technology can be used to screen significant proteins of differential expression in the serum of pancreatic cancer patients. These different proteins could be specific biomarkers of the patients with pancreatic cancer in the serum and have the potential value of further investigation.

Differential protein expression in rat cortical astrocytes following fluid percussion injury Two-dimensional gel electrophoresis and mass-spectrum detection

BACKGROUND： The Glasgow Coma Scale, computer tomography, and nuclear magnetic resonance imaging have been frequently used to diagnose brain injury. However, these methods do not accurately and quantitatively evaluate injury degree. However, proteomics displays some advantages. To date, there are few proteomics studies based on primary astrocyte cultures from a fluid percussion injury model. OBJECTIVE： To detect differential protein expression in rat cerebral cortical astrocytes following fluid percussion injury using two-dimensional gel electrophoresis and mass spectrum and to determine specific biological markers of brain injury. DESIGN, TIME AND SETTING： Complete, randomized grouping and proteomics experiments were performed at the Molecular Pathological Laboratory, Central Laboratory and Tianjin Key Laboratory for Biomarkers of Occupational and Environmental Hazard of Medical College of Chinese People＇s Armed Police Force from October 2007 to May 2008. MATERIALS： Inverted phase-contrast microscope was purchased from Olympus, Japan. PROTEAN IEF Cell isoelectric focusing electrophoresis system and PROTEAN II Xi-Cell vertical electrophoresis system were purchased from Bio-Rad, USA. Autofiex MALDI-TOF mass spectrometer was purchased from Bruker, Germany. METHODS： A total of 90 culture dishes, fully coated with Sprague Dawley rat cortical astrocytes, were randomly divided into control （n = 30） and injury （n = 60） groups. Astrocytes in the injury group were subjected to fluid percussion and subdivided into 4-hour （n = 30） and 48-hour injury （n = 30） groups. MAIN OUTCOME MEASURES： Cell morphology was observed using inverted phase-contrast microscopy. Cell total protein was extracted from each group, followed by two-dimensional gel electrophoresis and silver staining, and the differential protein expression was analyzed using PDQuest 7.0 software. Protein peptide mass fingerprinting of differential protein spots was obtained by matrix assisted laser desorption/ionization-time of flight mass spectrometry. The National Center for Biotechnology Information （NCBI） protein database was retrieved by Mascot to primarily identify protein type, Finally, differential protein expression was detected by Western blot analysis. RESULTS： Following fluid percussion injury, astrocytes displayed obvious swelling and increased intercellular space, with some cell detachment; the number of dead cells was significantly greater than the control group （P 〈 0.05）. Expression intensity of 114 protein spots was significantly greater in the injury group compared with the control group （P〈 0.05）; 9 of the 114 protein spots were identified and peptJde matching scores of 8 spots were 〉 61 （P 〈 0.05）. Protein types were identified and included cellular retinol binding protein, brain fatty acid binding protein 7, $100 calcium binding protein All, 60S acidic ribosomal protein P2, calponin 3, breast carcinoma amplified sequence 2 homolog, eukaryotic translation initiation factor 1A, and hypothetical protein LOC685814. Western blot detection revealed brain fatty acid binding protein 7 expression in cortical astrocytes, which increased with injury time compared with the control group （P 〈 0.05）. CONCLUSION： Results from this study showed morphological and proteomic changes in cortical astrocytes following fluid percussion injury. Brain fatty acid binding protein 7 was expressed in astrocytes and possibly played an important role in injury repair. Mass-spectrum identified differentially expressed proteins that correlated with cell metabolism regulation, signal transduction, and translation initiation, and could serve as specific biological markers of brain injury.