Biomarker is an important indicator that measures the events occurring in a biological system. Apart from being used as a sign to disclose the health status of an individual, biomarker is extremely useful in both toxicological research as well as biological monitoring. In addition to the occurrence of natural disease, exposure to toxic or specific environments may lead to fatal alteration of the biological system of a subject. However, if such alteration can be detected in the early stage, life will be spared upon appropriate medication. As a consequence, the evaluation of biomarker is extremely useful in the prevention of mortality.

Substances which cause renal damage are defined as nephrotoxic. Aristolochic acid (AA) is one of the examples of nephrotoxic substances. Research studie...[ Read more ]

Biomarker is an important indicator that measures the events occurring in a biological system. Apart from being used as a sign to disclose the health status of an individual, biomarker is extremely useful in both toxicological research as well as biological monitoring. In addition to the occurrence of natural disease, exposure to toxic or specific environments may lead to fatal alteration of the biological system of a subject. However, if such alteration can be detected in the early stage, life will be spared upon appropriate medication. As a consequence, the evaluation of biomarker is extremely useful in the prevention of mortality.

Substances which cause renal damage are defined as nephrotoxic. Aristolochic acid (AA) is one of the examples of nephrotoxic substances. Research studies and literature reveal that AA is strongly associated with the cases of renal fibrosis reported during the late 80s. Evidence has shown that AA will covalently bind to DNA to form stable adducts with remarkable persistence which can be detected after a reasonable long period of time. However, conventional analyses of DNA-AA adducts involve both tedious and invasive DNA isolation procedures in target organ. Herein, we report a non-invasive approach to detect and quantify DNA-AA adducts in rat urine rather than in tissue samples. The developed method entails extraction and enrichment of DNA-AA adducts in urine samples by solid phase extraction, followed by detection with the assistance of liquid chromatography-tandem mass spectrometry (LC-MS/MS). Since the newly developed method is to replace the old and traditional invasive method for adducts detection, method comparison is performed so as to prove that the developed method is of advantageous.

Apart from DNA, emerging evidence showed that RNA will be attacked under the influence of carcinogens. The impact is similar to that of DNA where the formation of covalent adducts may be resulted. However, since RNA does not possess proofreading enzyme such as DNA polymerase, there is no repair system for the elimination of the covalent adducts. Instead, RNA relies on enzymatic degradation or decay. Hence, RNA adducts may be released as free ribonucleosides and excreted through urinary system. As a result, we use an approach which is similar to DNA to detect the RNA-AA adducts in urine samples by UPLC-MS/MS. On the other hand, since the synthesis of proteins heavily relies on the integrity of RNA, we have a hypothesis that the deviation in RNA sequence will ultimately lead to mis-folding or truncation of proteins, therefore resulting in AA associated diseases such as kidney fibrosis.

Comparing to the traditional detection methods, the advantages of our developed method by utilizing mass spectrometry detection include high sensitivity and good selectivity. Identities of adducts have been made more promising and quantities of adducts are accurately evaluated. Although the traditional methods are interchangeable with our newly developed method, the enzymatic treatment of the old methods is still vital for other research studies. Thus, we take advantage on the natural occurrence of a DNA process called depurination: by hydrolysis with diluted hydrochloric acid, the covalent adducts from the DNA backbone will be released as free nucleobase adducts which are instrumentally detectable, saving the time for enzymatic hydrolysis. The acid hydrolysis method (mimicking the DNA depurination) shows that its performance is almost the same as the enzymatic hydrolysis for AA adducts, and it gives 80% efficiency on the other non-AA adducts within a much shorter period of time than the enzymatic hydrolysis method. The results are satisfactory which indicate that this method can be used to both detect and quantify the covalent adducts from DNA in the absence of enzyme. Quantification of DNA adducts thus becomes fast and simple.

With the developed methods, it is anticipated that the methods will be beneficial to both scientific and clinical research studies.