AA-DNA Adduct Research Articles

Aristolochic Acid Exposure via Dermal Contact or Inhalation of Herbal Powders: Evidence of Occupational Exposure in Herbalists with Urothelial Cancer.

Emerging evidence showing urothelial cancer in herbalists is linked to aristolochic acid (AA) exposure; however, the exposure pathway remains unclear. Here, we show that dermal contact and inhalation of fine powders of AA-containing herbs are significant occupational AA exposure pathways for herbalists. We initiated the study by quantifying the amount of AA in the AA-containing powder deposited on gloves and face masks worn by the operators of an AA-containing herb grinding machine. Then, we measured the kinetics of dermal absorption and dissolution of AA from fine powders of AA-containing herbs into artificial sweat and surrogate lung fluid. Lastly, we quantified the mutagenic AA-DNA adduct levels formed in the kidneys of mice exposed to AA-containing fine powders through dermal contact. Our findings highlight an urgent occupational risk that should demand implementation of safety standards for herbalists exposed to AA-containing fine powders.

Skullcapflavone II, a novel NQO1 inhibitor, alleviates aristolochic acid I-induced liver and kidney injury in mice.

Aristolochic acid I (AAI) is a well established nephrotoxin and human carcinogen. Cytosolic NAD(P)H quinone oxidoreductase 1 (NQO1) plays an important role in the nitro reduction of aristolochic acids, leading to production of aristoloactam and AA-DNA adduct. Application of a potent NQO1 inhibitor dicoumarol is limited by its life-threatening side effect as an anticoagulant and the subsequent hemorrhagic complications. As traditional medicines containing AAI remain available in the market, novel NQO1 inhibitors are urgently needed to attenuate the toxicity of AAI exposure. In this study, we employed comprehensive 2D NQO1 biochromatography to screen candidate compounds that could bind with NQO1 protein. Four compounds, i.e., skullcapflavone II (SFII), oroxylin A, wogonin and tectochrysin were screened out from Scutellaria baicalensis. Among them, SFII was the most promising NQO1 inhibitor with a binding affinity (KD = 4.198 μmol/L) and inhibitory activity (IC50 = 2.87 μmol/L). In human normal liver cell line (L02) and human renal proximal tubular epithelial cell line (HK-2), SFII significantly alleviated AAI-induced DNA damage and apoptosis. In adult mice, oral administration of SFII dose-dependently ameliorated AAI-induced renal fibrosis and dysfunction. In infant mice, oral administration of SFII suppressed AAI-induced hepatocellular carcinoma initiation. Moreover, administration of SFII did not affect the coagulation function in short term in adult mice. In conclusion, SFII has been identified as a novel NQO1 inhibitor that might impede the risk of AAI to kidney and liver without obvious side effect.

Co-Exposure to Aristolochic Acids I and II Increases DNA Adduct Formation Responsible for Aristolochic Acid I-Mediated Carcinogenicity in Rats.

The plant extract aristolochic acid (AA), containing aristolochic acids I (AAI) and II (AAII) as major components, causes aristolochic acid nephropathy (AAN) and Balkan endemic nephropathy (BEN), unique renal diseases associated with upper urothelial cancer. Recently (Chemical Research in Toxicology 33(11), 2804–2818, 2020), we showed that the in vivo metabolism of AAI and AAII in Wistar rats is influenced by their co-exposure (i.e., AAI/AAII mixture). Using the same rat model, we investigated how exposure to the AAI/AAII mixture can influence AAI and AAII DNA adduct formation (i.e., AA-mediated genotoxicity). Using 32P-postlabelling, we found that AA-DNA adduct formation was increased in the livers and kidneys of rats treated with AAI/AAII mixture compared to rats treated with AAI or AAII alone. Measuring the activity of enzymes involved in AA metabolism, we showed that enhanced AA-DNA adduct formation might be caused partially by both decreased AAI detoxification as a result of hepatic CYP2C11 inhibition during treatment with AAI/AAII mixture and by hepatic or renal NQO1 induction, the key enzyme predominantly activating AA to DNA adducts. Moreover, our results indicate that AAII might act as an inhibitor of AAI detoxification in vivo. Consequently, higher amounts of AAI might remain in liver and kidney tissues, which can be reductively activated, resulting in enhanced AAI DNA adduct formation. Collectively, these results indicate that AAII present in the plant extract AA enhances the genotoxic properties of AAI (i.e., AAI DNA adduct formation). As patients suffering from AAN and BEN are always exposed to the plant extract (i.e., AAI/AAII mixture), our findings are crucial to better understanding host factors critical for AAN- and BEN-associated urothelial malignancy.

The influence of ochratoxin A on DNA adduct formation by the carcinogen aristolochic acid in rats.

quinone oxidoreductase (NQO1) activating AA to DNA adducts, their combined treatment did not lead to either higher NQO1 enzyme activity or higher AA-DNA adduct levels in ex vivo incubations. Oxidation of AA I (8-methoxy-6-nitrophenanthro[3,4-d]-1,3-dioxole-5-carboxylic acid) to its detoxification metabolite, 8-hydroxyaristolochic acid, was lower in microsomes from rats treated with AA and OTA, and this was paralleled by lower activities of cytochromes P450 1A1/2 and/or 2C11 in these microsomes. Our results indicate that a decrease in AA detoxification after combined exposure to AA and OTA leads to an increase in AA-DNA adduct formation in liver and kidney of rats.

Mutagenicity and DNA adduct formation by aristolochic acid in the spleen of Big Blue® rats.

Aristolochic acid (AA) is a potent human nephrotoxin and carcinogen. We previously reported that AA treatment resulted in DNA damage and mutation in the kidney and liver of rats. In this study, we have determined the DNA adducts and mutations induced by AA in rat spleen. Big Blue® transgenic rats were gavaged with 0, 0.1, 1.0, and 10.0 mg AA/kg body weight five-times/week for 3 months. Three DNA adducts, [7-(deoxyadenosin-N(6)-yl)-aristolactam I, 7-(deoxyadenosin-N(6)-yl)-aristolactam II and 7-(deoxyguanosin-N(2)-yl)-aristolactam I], were identified by (32)P-postlabeling. Over the dose range studied, there were strong linear dose-responses for AA-DNA adduct formation in the treated rat spleens, ranging from 4.6 to 217.6 adducts/10(8) nucleotides. Spleen cII mutant frequencies also increased in a dose-dependent manner, ranging from 32.7 to 286.2 × 10(-6) in the treated animals. Mutants isolated from the different treatment groups were sequenced; analysis of the resulting spectra indicated that there was a significant difference between the pattern of mutation in the 10 mg/kg AA-treated and the vehicle control rats. A:T → T:A transversion was the major type of mutation in AA-treated rats, whereas G:C → A:T transition was the main type of mutation in the vehicle controls. These results indicate that AA is genotoxic in the spleen of rats exposed under conditions that result in DNA adduct formation and mutation induction in kidney and liver.

Ochratoxin A and Aristolochic Acid Involvement in Nephropathies and Associated Urothelial Tract Tumours

This review addresses the unresolved aetiology of several nephropathies and associated upper tract tumours diagnosed all over the world, but especially in the Balkan regions. Studies conducted over the last 35 years point to mycotoxins, mainly ochratoxin A (OTA) as the main culprit. Recent theories however have implicated aristolochic acids (AA). The aim of this review is to put forward arguments in favour of the mycotoxin theory and to show the incoherence of the AA theory. It discusses the differences between the epidemiology of Balkan endemic nephropathy (BEN) and aristolochic acid nephropathy (AAN); OTA and AA carcinogenicity; clinical and pathological effects induced by OTA and AA; sources of OTA contamination (food, air, drinking water); OTA- and AA-DNA adduct formation; the role of genetic polymorphisms; and the risk for young children.

Improved preparation and identification of aristolochic acid-DNA adducts by solid-phase extraction with liquid chromatography-tandem mass spectrometry

Aristolochic acid (AA) is a known nephrotoxin and potential carcinogen, which can form covalent DNA adducts after metabolic activation in vivo and in vitro. A simple method for preparation and characterization of aristolochic acid-DNA adducts was developed. Four AA-adducts were synthesized by a direct reaction of AAI/AAII with 2'-deoxynucleosides. The reaction mixture was first cleaned-up and pre-concentrated using solid phase extraction (SPE), and further purified by a reversed-phase high performance liquid chromatography (HPLC). By the application of developed SPE procedure, matrices and byproducts in reaction mixture could be greatly reduced and adducts of high purity (more than 94% as indicated by HPLC) were obtained. The purified AA-DNA adducts were identified and characterized with liquid-electrospray ionization-quadrupole-time of flight-mass spectrometry (LC-ESI-Q-TOF-MS/MS) and LC-Diode array detector-fluorescence (LC-DAD-FL) analysis. This work provides a robust tool for possible large-scale preparation of AA-DNA adduct standards, which can promote the further studies on carcinogenic and mutagenic mechanism of aristolochic acids.

New molecular and field evidences for the implication of mycotoxins but not aristolochic acid in human nephropathy and urinary tract tumor

To find out whether ochratoxin A (OTA), citrinin (CIT), aristolochic acids (AA) are etiologic agents of Balkan endemic nephropathy (BEN) or Chinese herbal nephrotoxicity, and associated urinary tract tumor (UTT), we have compared (i) in human kidney cell culture, the DNA adduct formation and persistence of OTA/CIT and AA adducts (ii) analyzed DNA adduct in several tumors from human kidney suspected to be exposed to either OTA and CIT, or AAs (iii) analyzed OTA, CIT, and AA in food. In kidney cell cultures, formation of specific OTA-DNA adduct and AA-DNA adduct were detected in the same range (around 10 adducts/10(9) nucleotides) and were time- and dose-dependent. After 2 days all disappeared. DNA adduct related to OTA and CIT are found in human kidney tissues from Balkans, France, and Belgium whereas no DNA adducts related to AA could be found in any tumors of BEN patients from Croatia, Bulgaria, or Serbia. No DNA adduct was found in kidney biopsy or necropsy of the French women suspected to be exposed to AA. OTA and CIT are more frequently found in rural area. AA was never detected. All these plead for implication of mycotoxins, especially OTA, in BEN and UTT.

Metabolic activation of carcinogenic aristolochic acid, a risk factor for Balkan endemic nephropathy

Aristolochic acid (AA), a naturally occurring nephrotoxin and carcinogen, is associated with tumor development in patients suffering from Chinese herbs nephropathy (now termed aristolochic acid nephropathy, AAN) and may also be a cause for the development of a similar type of nephropathy, the Balkan endemic nephropathy (BEN). Major DNA adducts [7-(deoxyadenosin-N6-yl)-aristolactam and 7-(deoxyguanosin-N2-yl)aristolactam] formed from AA after reductive metabolic activation were found in renal tissues of patients with both diseases. Understanding which human enzymes are involved in AA activation and/or detoxication is important in the assessment of an individual's susceptibility to this plant carcinogen. This paper reviews major hepatic and renal enzymes responsible for AA-DNA adduct formation in humans. Phase I biotransformation enzymes play a crucial role in the metabolic activation of AA to species forming DNA adducts, while a role of phase II enzymes in this process is questionable. Most of the activation of AA in human hepatic microsomes is mediated by cytochrome P450 (CYP) 1A2 and, to a lower extent, by CYP1A1; NADPH:CYP reductase plays a minor role. In human renal microsomes NADPH:CYP reductase is more effective in AA activation. Prostaglandin H synthase (cyclooxygenase, COX) is another enzyme activating AA in human renal microsomes. Among the cytosolic reductases, NAD(P)H:quinone oxidoreductase (NQO1) is the most efficient in the activation of AA in human liver and kidney. Studies with purified enzymes confirmed the importance of CYPs, NADPH:CYP reductase, COX and NQO1 in the AA activation. The orientation of AA in the active sites of human CYP1A1, -1A2 and NQO1 was predicted from molecular modeling and explains the strong reductive potential of these enzymes for AA detected experimentally. We hypothesized that inter-individual variations in expressions and activities of enzymes activating AA may be one of the causes responsible for the different susceptibilities to this carcinogen reflected in the development of AA-induced nephropathies and associated urothelial cancer.

Early proximal tubule injury in experimental aristolochic acid nephropathy: functional and histological studies

Aristolochic acid (AA), the plant extract of Aristolochia species, is involved in the onset of progressive tubulointerstitial renal fibrosis in humans. Clinical and in vitro findings have previously suggested that the proximal tubule was the target of AA. Using a rat model of AA nephropathy, the proximal tubular lesions induced by daily subcutaneous injections of AA for 35 or 5 days were characterized biochemically and histologically. Urinary excretion of proteins, albumin, low molecular weight proteins, N-acetyl-beta-d-glucosaminidase, alpha-glutathione S-transferase, leucine aminopeptidase and neutral endopeptidase (NEP) was determined and related to histological conventional findings and immunostainings of NEP and megalin. In both protocols, an acute phase of release of urinary markers was observed within the first 3 days of AA treatment in parallel with a significant increase of specific AA-related DNA adducts reflecting early tubular intoxication. A dramatic loss of the proximal tubule brush border was histologically confirmed, while the expression of megalin decreased at the damaged apical epithelium (mainly of the S3 segment). Proximal tubule injury occurs early after AA intoxication in rats, with a link between specific AA-DNA adduct formation, decreased megalin expression and inhibition of receptor-mediated endocytosis of low molecular weight proteins, bringing in vivo confirmation of previous in vitro studies.

Effects of dexfenfluramine on aristolochic acid nephrotoxicity in a rat model for Chinese-herb nephropathy

Chinese-herb nephropathy (CHN) is a progressive renal interstitial fibrosis initially reported after concomitant intake of an anorexigen, (dex)fenfluramine, and a Chinese herb ( Aristolochia fangchi) containing nephrotoxic and carcinogenic aristolochic acid (AA). We thus tested the possible enhancing effect of the active enantiomer dexfenfluramine (DXF) on AA nephrotoxicity in a rat model for CHN. Groups of 12 salt-depleted male Wistar rats received daily subcutaneous injections of 7 mg/kg body weight DXF (DXF group), 7 mg/kg body weight AA (AA group), a combination of the same doses of AA and DXF (AA+DXF group), or vehicle (control group) for up to 35 days. Six animals per group were killed on day 10 and the remaining six on day 35. Renal function was evaluated by determining serum creatinine and urinary leucine aminopeptidase activity. Histological evaluation of kidney samples was performed and tubulointerstitial injuries were semiquantified. The DXF group did not differ from controls for any parameter. Similarly elevated serum creatinine levels, decreased leucine aminopeptidase enzymuria, and renal lesions were observed in the AA and the AA+DXF groups after both 10 and 35 days. The formation of specific AA-DNA adducts in liver and renal tissue samples was assessed by the (32)P-postlabelling method. Specific AA-DNA adduct levels were significantly increased in kidney tissues from AA+DXF rats compared with AA rats. These functional and histological data suggest that DXF does not enhance AA nephrotoxicity in a rat model for CHN. Further investigations are needed to clarify the mechanism by which DXF may enhance AA-DNA adduct formation.

Carcinogenic aristolochic acids upon activation by DT-diaphorase form adducts found in DNA of patients with Chinese herbs nephropathy.

Aristolochic acid (AA), a naturally occurring nephrotoxin and rodent carcinogen, has recently been associated with the development of urothelial cancer in humans. Understanding which enzymes are involved in AA activation and/or detoxication is important in the assessment of an individual susceptibility to this natural carcinogen. We examined the ability of enzymes of rat renal and hepatic cytosolic fractions to activate AA to metabolites forming DNA adducts by the nuclease P1-enhanced version of the (32)P-postlabeling assay. Cytosolic fractions of both these organs generated AA-DNA adduct patterns reproducing those found in renal tissues from humans exposed to AA. 7-(Deoxyadenosin-N(6)-yl)aristolactam I, 7-(deoxyguanosin-N(2)-yl)aristolactam I and 7-(deoxyadenosin-N(6)-yl)aristolactam II were identified as AA-DNA adducts formed from AAI and 7-(deoxyguanosin-N(2)-yl)aristolactam II and 7-(deoxyadenosin-N(6)-yl)aristolactam II were generated from AAII by hepatic cytosol. Qualitatively the same AA-DNA adduct patterns were observed, although at lower levels, upon incubation of AAs with renal cytosol. To define the role of cytosolic reductases in the reductive activation of AA, we investigated the modulation of AA-DNA adduct formation by cofactors, specific inducers or selective inhibitors of the cytosolic reductases, DT-diaphorase, xanthine oxidase (XO) and aldehyde oxidase. The role of the enzymes in AA activation was also investigated by correlating the DT-diaphorase- and XO-dependent catalytic activities in cytosolic sample with the levels of AA-DNA adducts formed by the same cytosolic sample. On the basis of these studies, we attribute most of the cytosolic activation of AA to DT-diaphorase, although a role of cytosolic XO cannot be ruled out. With purified DT-diaphorase, the participation of this enzyme in the formation of AA-DNA adducts was confirmed. The binding orientation of AAI in the active site of DT-diaphorase was predicted by computer modeling based on published X-ray structures. The results presented here are the first report demonstrating a reductive activation of carcinogenic AAs by DT-diaphorase.

Human enzymes involved in the metabolic activation of carcinogenic aristolochic acids: evidence for reductive activation by cytochromes P450 1A1 and 1A2.

Aristolochic acid (AA), a naturally occurring nephrotoxin and rodent carcinogen, has recently been associated with the development of urothelial cancer in humans. Determining the capability of humans to metabolize AA and understanding, which human enzymes are involved in AA activation is important in the assessment of individual susceptibility. Using the nuclease P1-enhanced version of the (32)P-postlabeling assay, we compared the ability of human, minipig and rat hepatic microsomal samples to activate AA to metabolites forming DNA adducts. Human microsomes generated AA-DNA adduct profiles reproducing those found in renal tissues from humans exposed to AA. Identical patterns of AA-DNA adducts were also observed when AA was activated by minipig and rat microsomes. Therefore, microsomes of both animals are suitable in vitro systems mimicking the enzymatic activation of AA in humans. To define the role of specific P450 enzymes and NADPH:P450 reductase in the activation of AA by human microsomes we investigated the modulation of AA-DNA adduct formation by specific inducers or selective inhibitors of P450s and cofactors or inhibitors of NADPH:P450 reductase. The inducer of P450 1A1/2, beta-naphthoflavone, significantly stimulated the levels of AA-DNA adducts formed by rat microsomes, but inducers of P450 2B1/2 and 2E1 had no such effect. Furthermore, only inhibitors of the P450 1A subfamily (alpha-naphthoflavone, furafylline) significantly decreased the amount of adducts formed by microsomes from humans, minipigs and rats. alpha-Lipoic acid, an inhibitor of NADPH:P450 reductase, inhibited adduct formation too, but to a lower extent. On the basis of these results, we attribute most of the microsomal activation of AA to P450 1A1 and 1A2, although a role of NADPH:P450 reductase cannot be ruled out. With purified enzymes (recombinant P450 1A1/2 and NADPH:P450 reductase) and microsomes from baculovirus transfected insect cells expressing recombinant human P450 1A1/2 and NADPH:P450 reductase, the participation of these enzymes in the formation of AA-DNA adducts was confirmed. These results are the first report on the activation of AA by human enzymes and clearly demonstrate the role of P450 1A1, 1A2, and NADPH:P450 reductase in catalyzing the reductive activation of AA.

Analyses of DNA adducts formed by ochratoxin A and aristolochic acid in patients with Chinese herbs nephropathy

Chinese herbs nephropathy (CHN), a unique type of nephropathy has been associated with the intake of weight-reducing pills containing the Chinese herb Aristolochia fangchi. Moreover, an association between the use of A. fangchi and urothelial cancer in CHN patients has been reported indicating that aristolochic acid (AA) the major alkaloid of A. fangchi might be the causal agent. Similarities of CHN to the Balkan endemic nephropathy (BEN) have led to the hypothesis of a common etiological agent for both diseases. Evidence has accumulated that BEN is an environmentally-induced disease strongly associated with the fungal mycotoxin ochratoxin A (OTA). Both, AA and OTA are nephrotoxic and carcinogenic and induce the formation of DNA adducts. As OTA has been suspected as fungal contaminant in the herbal batches used for the preparation of the weight-reducing pills we analysed tissues from CHN patients by the 32 P -postlabeling procedure for the presence of DNA adducts related to both OTA and AA exposure. Whereas, AA-specific DNA adducts were detected in all five urinary tract tissues from five patients (total RAL: 32–251 adducts per 10 9 nucleotides), OTA-related DNA adducts were detectable in two kidneys and one ureter only (total RAL: 1.5–3.7 adducts per 10 9 nucleotides). Thus, OTA-related DNA adduct levels were about 50 times lower than AA-DNA adduct levels. In female and male rats that were treated with the slimming regimen in the same way like the CHN patients except that the amount of Chinese herbs was 10 times higher, AA-DNA adducts were found in kidney tissues (total RAL ranging from 51 to 83 adducts per 10 9 nucleotides) but adducts derived from OTA were not observed. These results demonstrate that OTA-related DNA adducts do not play a key role in CHN or CHN-associated urothelial cancer.

Evidence for reductive activation of carcinogenic aristolochic acids by prostaglandin H synthase — 32P-postlabeling analysis of DNA adduct formation

Aristolochic acid (AA), a naturally occurring nephrotoxin and carcinogen, is implicated in an unique type of renal fibrosis, designated Chinese herbs nephropathy (CHN), which can develop to urothelial cancer. Understanding which enzymes are involved in AA activation and/or detoxication is important in the assessment of an individual susceptibility to this natural carcinogen. We examined the ability of prostaglandin H synthase (PHS) to activate AA to metabolites forming DNA adducts with the nuclease P1 and 1-butanol extraction enrichment procedure of the 32P-postlabeling assay. PHS is a prominent enzyme in the kidney and urothelial tissues. Ram seminal vesicle (RSV) microsomes, which contain high levels of PHS, generated AA-DNA adduct patterns reproducing those found in renal tissues in CHN patients. 7-(Deoxyadenosin- N 6-yl)aristolactam I, 7-(deoxyguanosin- N 2-yl)aristolactam I and 7-(deoxyadenosin- N 6-yl)aristolactam II were identified as AA-DNA adducts formed by AAI. Two adducts, 7-(deoxyguanosin- N 2-yl)aristolactam II and 7-(deoxyadenosin- N 6-yl)aristolactam II, were generated from AAII. According to the structures of the DNA adducts identified, nitroreduction is the crucial pathway in the metabolic activation of AA. The identity of PHS as the activating enzyme in RSV microsomes was proven with different cofactors and inhibitors. Only indomethacin, a selective inhibitor of PHS, significantly decreased the amount of adducts formed by RSV microsomes. The inhibitor of NADPH:CYP reductase (α-lipoic acid) and some selective inhibitors of cytochromes P450 (CYP) were not effective. Likewise, only cofactors of PHS, arachidonic acid and hydrogen peroxide, supported the DNA adduct formation of AAI and AAII, while NADPH and NADH were ineffective. These results demonstrate a key role of PHS in the activation pathway of AAI and AAII in the RSV microsomal system and were corroborated with the purified enzyme, namely ovine PHS-1. The results presented here are the first report demonstrating a reductive activation of nitroaromatic compounds by PHS-1.