Effects of Heavy Metal Co-Exposure on the formation of DNA Adducts from Aristolochic Acid I: Implications for Balkan Endemic Nephropathy Development.

Accumulated evidence has shown that Balkan endemic nephropathy (BEN) is a multifactorial environmental disease, with exposure to aristolochic acids (AA), and the associated DNA adduct formation, as a key causative factor of BEN development. Here, we show that coexposure to arsenic, cadmium, and iron increases the DNA adduct formation of AA in cultured kidney cells, while exhibiting both an exposure concentration and duration dependence. In contrast, coexposure to calcium and copper showed a decreasing DNA adduct formation. Because DNA damage is responsible for both the nephrotoxicity and carcinogenicity of AA, these results shed greater light on the endemic nature of BEN.

Fabrication of a Simple and Efficient HPLC Reduction Column for Online Conversion of Aristolochic Acids to Aristolactams Prior to Sensitive Fluorescence Detection.

Aristolochic acids (AAs) are nephrotoxic and carcinogenic nitrophenanthrene carboxylic acids produced naturally by plants from the Aristolochia and Asarum genera, which have been used extensively as herbal medicines. In addition to consuming AA-containing herbal medicinal products, there is emerging evidence that humans are also exposed to AA through the environment. In 2022, the World Health Organization (WHO) called for global action to remove AA exposure sources and to implement preventative measures against the development of AA-associated cancers. Herein, we report the development of a simple and efficient iron powder-packed reduction column that allows online post-column conversion of the nonfluorescing AA to its corresponding strongly fluorescing aristolactam (AL), facilitating the sensitive and selective detection of AA in herbal medicinal products, food grain, arable soil, or groundwater samples by high-performance liquid chromatography with fluorescence detection. Moreover, AL, a group of naturally occurring derivatives of AA that have demonstrated toxicity to cultured bacteria, human cells, and rats, is monitored and quantified simultaneously with AA in one single run without sacrificing sensitivity. In comparison with existing analytical methods for AA measurement, the newly developed method is not only inexpensive and less laborious, but it also offers improved sensitivity. We believe this novel method will find wide application in identifying the presence of AA in food, herbal medicines, and environmental samples, thus assisting in the identification and removal of AA exposure sources.

Occurrence and stability of PCMX in water environments and its removal by municipal wastewater treatment processes.

Para-chloro-meta-xylenol (PCMX) is a synthetic antiseptic used extensively to control the spread of germs and viruses, and as a result, enormous amount of PCMX could be discharged to water environments through drainage. To investigate the extent of PCMX contamination, water samples were collected from rivers and coastal waters in Hong Kong, and PCMX concentrations were determined by a newly developed method using liquid chromatography-tandem mass spectrometry combined with stable isotope-dilution. We discovered widespread PCMX pollution in the water environment. Then, we revealed for the first time that PCMX in wastewater is not effectively removed by chemically enhanced primary treatment (CEPT), one of the wastewater treatment processes used in Hong Kong (∼75% of wastewater) and other megacities around the world. This suggests that the CEPT effluent or the primary treatment effluent is an unintended continuous source of pollution for PCMX in water environments. Finally, we found that PCMX was relatively stable in the water environment and could pose a risk to aquatic organisms. These findings underscore the importance of raising public awareness of the environmental consequences from overuse of PCMX-based disinfectants and the need to reevaluate the various wastewater treatment processes in removing PCMX.

Bioaccumulation and DNA Adduct Formation of Aristolactam I: Unmasking a Toxicological Mechanism in the Pathophysiology of Aristolochic Acid Nephropathy.

Prolonged exposure to aristolochic acid (AA) through AA-containing herbal medicines or AA-tainted food is putting a large portion of the global population at risk of developing renal fibrosis and tumors of the upper urinary tract. In an effort to better understand the organotropic property of AA, we studied the cytotoxicity, absorption, oxidative-stress inducing potential, and DNA adduct formation capability of aristolactam I (ALI), one of the major urinary metabolites of aristolochic acid I (AAI) in human cells. Despite ALI having a slightly lower cytotoxicity than that of AAI, the analysis revealed, for the first time, that ALI is bioaccumulated 900 times more than that of AAI inside cultured kidney cells. Furthermore, ALI induced a significantly larger glutathione depletion than that of AAI in the exposed cells. Together with the formation of ALI-DNA adduct at a reasonably high abundance, results of this study unmasked a previously disregarded causative role of ALI in the organotropic tumor-targeting property of AA.

Determination of Aristolochic Acids in Vegetables: Nephrotoxic and Carcinogenic Environmental Pollutants Contaminating a Broad Swath of the Food Supply and Driving Incidence of Balkan Endemic Nephropathy.

Balkan endemic nephropathy (BEN) is a slowly progressive interstitial fibrotic disease affecting numerous people living along the Danube River in the Balkan Peninsula, of which aristolochic acids (AAs) produced naturally in Aristolochia plants are key etiological agents. However, the exposure biology of the disease remains poorly understood. Initially, the high incidence of BEN in the Balkan Peninsula was thought to occur through ingestion of bread prepared from flour made with wheat grains comingled with the seeds of Aristolochia clematitis L., an AA-containing weed that grows abundantly in the wheat fields of the affected areas. In this study, by a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, we show for the first time that vegetables, in particular root vegetables of endemic areas, are extensively contaminated with AAs taken up through root absorption from the AA-tainted soil. Furthermore, we found a pH dependence of the n-octanol/water partition coefficient (Kow) of AAs, which resulted in a dramatically higher hydrophobicity-driven plant uptake efficiency of AAs into food crops in endemic areas, characterized by higher acidity levels, compared to non-endemic areas. We believe the results of this study have significantly unraveled the mystery surrounding the uneven distribution of BEN incidence.

Quantitation of DNA Adducts of Aristolochic Acids in Repair-Deficient Cells: A Mechanistic Study of the DNA Repair Mechanism.

Accumulating evidence has revealed that nephrotoxic and carcinogenic aristolochic acids (AAs) released from decaying Aristolochia clematitis L. weeds are soil and food grain contaminants in the Balkan Peninsula, while AA toxicity has been linked to induced DNA damage. In this study, we investigated the DNA repair mechanism that excises the aristolactam-DNA adducts in gene-knockout Escherichia coli cells. These results demonstrated that cell lines deficient in nucleotide excision repair (NER) machinery accumulated higher adduct levels, indicating that NER is the major mechanism responsible for the repair of these lesions. Furthermore, data also revealed the involvement of base excision repair enzymes in repairing the lesions but with lower contribution than NER.