[Risk assessment, safe medication and scientific supervision of traditional Chinese medicine containing aristolochic acids--toxicity is different among aristolochic acids, and detection and control of aristolochic acid â /â ¡ is critical].

The safety problem of traditional Chinese medicine containing aristolochic acid is of great concern in China and abraod, which poses a challenge in clinical application and supervision. There are many types of aristolochic acid analogues(AAAs) and 178 have been reported. According to the structure, they are classified into aristolochic acids(AAs) and aristololactams(ALs). The toxi-city is remarkably different among AAAs of different types. For example, AA-Ⅰ has strong nephrotoxicity and carcinogenicity, and the toxicity of AA-Ⅱ is lower than that of AA-Ⅰ. Besides, AA-Ⅳa and AA-Ⅰa are considered to have no obvious nephrotoxicity and carcinogenicity. The types and content of AAAs are significantly different among traditional Chinese medicines derived from different Aristolochiaceae species. For example, Asari Radix et Rhizoma and Aristolochiae Herba mainly consist of AAAs without obvious toxicity(such as AA-Ⅳa). The content of AAAs in compound preparations is related to the proportions of the medicinals and the processing method. The content of AA-Ⅰ in some compound preparations is very low or below the detection limit. Therefore, the author concludes that AAAs of different types have different toxicity, but not all AAAs has nephrotoxicity and carcinogenicity. Moreover, the toxicity of traditional Chinese medicines containing AAAs should not be generalized and AA-Ⅰ and AA-Ⅱ should be emphasized. In this paper, it is suggested that traditional Chinese medicine containing AAAs should be used rationally and research, analysis, and toxicological study of AAAs species and content should be strengthened. In addition, limit standards of AA-Ⅰ and AA-Ⅱ should be formulated and science-based supervision should be performed.

Pig-a gene mutation assay study design: critical assessment of 3- versus 28-day repeat-dose treatment schedules.

The in vivo Pig-a assay is being used in safety studies to evaluate the potential of chemicals to induce somatic cell gene mutations. Ongoing work is aimed at developing an Organisation for Economic Cooperation and Development (OECD) test guideline to support routine use for regulatory purposes (OECD project number 4.93). Among the details that will need to be articulated in an eventual guideline are recommended treatment and harvest schedules. With this in mind, experiments reported herein were performed with Wistar Han rats exposed to aristolochic acid I (AA), 1,3-propane sultone, chlorambucil, thiotepa or melphalan using each of two commonly used treatment schedules: 3 or 28 consecutive days. In the case of the 3-day studies, blood was collected for Pig-a analysis on days 15 or 16 and 29 or 30. For the 28-day studies blood was collected on day 29 or 30. The effect of treatment on mutant reticulocytes and mutant erythrocytes was evaluated with parametric pair-wise tests. While each of the five mutagens increased mutant phenotype cell frequencies irrespective of study design, statistical significance was consistently achieved at lower dose levels when the 28-day format was used (e.g. 2.75 vs 20 mg/kg/bw for AA). To more thoroughly investigate the dose-response relationships, benchmark dose (BMD) analyses were performed with PROAST software. These results corroborate the pair-wise testing results in that lower BMD values were obtained with the 28-day design. Finally, mutagenic potency, as measured by BMD analyses, most consistently correlated with the mutagens' tumorigenic dose 50 values when the lengthier treatment schedule was used. Collectively, these results suggest that both 3- and 28-day treatment schedules have merit in hazard identification-type studies. That being said, for the purpose of regulatory safety assessments, there are clear advantages to study designs that utilise protracted exposures.

Assessment of Pig-a, Micronucleus, and Comet Assay Endpoints in Tg.RasH2 Mice Carcinogenicity Study of Aristolochic Acid I.

A newly developed in vivo Pig-a gene mutation assay displays great potential for integration into genotoxicity tests. To obtain more evidence for application of the Pig-a assay, we integrated this assay, micronucleus test in peripheral blood (MN-pb test) and bone marrow (MN-bm test), as well as a Comet assay into a transgenic RasH2 mice carcinogenicity study. Fourteen male RasH2 mice and five wild-type (WT) mice were treated with a strong mutagen aristolochic acid I at a dose of 5 mg/kg/day for 4 consecutive weeks. Mice recovered in 5 weeks. Peripheral bloods were collected for Pig-a assay, MN-pb test, and Comet assay at several time points, while bone marrow and target organs were harvested for the MN-bm test and pathological diagnosis after mice were euthanized. Finally, 13 of the 14 RasH2 mice developed squamous cell carcinomas in the forestomach, while there were no carcinomas in the WT mice. Pig-a mutant frequencies (MFs) consecutively increased throughout the study to a maximum value of approximately 63-fold more than background. These frequencies were relative to the incidence, size, and malignant degree of tumors. Micronucleated reticulocytes increased from Day 1 to Day 49, before returning to background levels. No positive responses were observed in either the MN-bm test or the Comet assay. Results suggested that, when compared with the other two tests, the Pig-a assay persistently contributed to sustaining MFs, enhanced detection sensitivity due to the accumulation of Pig-a mutations, and demonstrated better predictability for tumorigenicity. Environ. Mol. Mutagen. 61:266-275, 2020. © 2019 Wiley Periodicals, Inc.

Assessment of nephrotoxicity of herbal medicine containing aristolochic acid in mice.

BACKGROUND/AIMS: It is undetermined if herbal medicines (HM) containing aristolochic acid (AA)-containing have similar nephrotoxicity to AA itself. METHODS: We administered HM containing a high concentration of AA for 5 days (short-term study) or a low concentration of AA for 30 days (long-term study) to C57BL/6 mice; for comparison, same dose of AA compound was used as controls. RESULTS: The nephrotoxicity in the HM- and AA-treated mice was compared in terms of renal function, histopathology, oxidative stress, apoptotic cell death, and mitochondrial damage. Short-term HM treatment resulted in acute kidney injury (marked renal dysfunction, acute tubular necrosis, and neutrophil gelatinase-associated lipocalin [NGAL] expression) in which the severity of renal dysfunction and histopathology was comparable with that induced by the administration of AA alone. Long-term HM treatment resulted in features of chronic kidney disease (CKD, mild renal dysfunction and tubular atrophy and dilatation). No significant differences in these parameters were observed between the HM- and AA-treated mice. HM-induced oxidative stress (8-hydroxy-2'-deoxyguanosine and manganese- dependent superoxide dismutase expression) and apoptotic cell death (terminal deoxynucleotidyl transferase dUTP nick end labelling [TUNEL]-positive cells and active caspase-3 expression) were similar in HM- and AA-treated mice in the short-term and long-term studies. Mitochondrial injury, evaluated by electron microscopy, was also similar in HM- and AA-treated mice in the short-term and long-term studies. CONCLUSION: The nephrotoxic potential of HM containing AA was similar to that of AA itself.

Premalignant alteration assessment in liver-like tissue derived from embryonic stem cells by aristolochic acid I exposure.

The in vitro predictive evaluation of chemical carcinogenicity based on hepatic premalignance has so far not been established. Here, we report a novel approach to investigate the premalignant events triggered by human carcinogen aristolochic acid I (AAI) in the liver-like tissue derived from mouse embryonic stem cells. By AAI exposure, the liver-like tissue exhibited the paracrine interleukin-6 phenotypic characteristics. Hepatocytes expressed STAT3/p-STAT3, c-Myc and Lin28B in parallel. Some of them displayed the dedifferentiation characteristics, such as full of α-fetoprotein granules, increase in size, and nucleocytoplasmic shuttle of Oct4. When these cells were injected into mice, the xenografts mostly displayed the uniform area of hepatic-like tissue with malignant nuclei. The hepatic malignant markers, α-fetoprotein, cytokeratin 7 and cytokeratin 19, were co-expressed in albumin-positive areas, respectively. In conclusion, we established an approach to predict the hepatic premalignance triggered by carcinogen AAI. This premalignant assay system might aid to evaluate the effects of potential carcinogens in liver, and probably to screen the protecting against hepatocarcinogenic efficacy of pharmaceuticals in vitro.

Predicting points of departure for risk assessment based on in vitro cytotoxicity data and physiologically based kinetic (PBK) modeling: The case of kidney toxicity induced by aristolochic acid I.

Aristolochic acids are naturally occurring nephrotoxins. This study aims to investigate whether physiologically based kinetic (PBK) model-based reverse dosimetry could convert in vitro concentration-response curves of aristolochic acid I (AAI) to in vivo dose response-curves for nephrotoxicity in rat, mouse and human. To achieve this extrapolation, PBK models were developed for AAI in these different species. Subsequently, concentration-response curves obtained from in vitro cytotoxicity models were translated to in vivo dose-response curves using PBK model-based reverse dosimetry. From the predicted in vivo dose-response curves, points of departure (PODs) for risk assessment could be derived. The PBK models elucidated species differences in the kinetics of AAI with the overall catalytic efficiency for metabolic conversion of AAI to aristolochic acid Ia (AAIa) being 2-fold higher for rat and 64-fold higher for mouse than human. Results show that the predicted PODs generally fall within the range of PODs derived from the available in vivo studies. This study provides proof of principle for a new method to predict a POD for in vivo nephrotoxicity by integrating in vitro toxicity testing with in silico PBK model-based reverse dosimetry.

Assessment of the role of renal organic anion transporters in drug-induced nephrotoxicity.

In the present review we have attempted to assess the involvement of the organic anion transporters OAT1, OAT2, OAT3, and OAT4, belonging to the SLC22 family of polyspecific carriers, in drug-induced renal damage in humans. We have focused on drugs with widely recognized nephrotoxic potential, which have previously been reported to interact with OAT family members, and whose underlying pathogenic mechanism suggests the participation of tubular transport. Thus, only compounds generally believed to cause kidney injury either by means of direct tubular toxicity or crystal nephropathy have been considered. For each drug, or class of agents, the evidence for actual transport mediated by individual OATs under in vivo conditions is discussed. We have then examined their role in the context of other carriers present in the renal proximal tubule sharing certain substrates with OATs, as these are critical determinants of the overall contribution of OAT-dependent transport to intracellular accumulation and transepithelial drug secretion, and thus the impact it may have in drug-induced nephrotoxicity.