Mutational Signature Analysis Reveals Widespread Contribution of Pyrrolizidine Alkaloid Exposure to Human Liver Cancer.

BACKGROUND AND AIMS: Mutational signature analyses are an effective tool in identifying cancer etiology. Humans are frequently exposed to pyrrolizidine alkaloids (PAs), the most common carcinogenic phytotoxins widely distributed in herbal remedies and foods. However, due to the lack of human epidemiological data, PAs are classified as group II hepatocarcinogens by the World Health Organization. This study identified a PA mutational signature as the biomarker to investigate the association of PA exposure with human liver cancer. APPROACH AND RESULTS: Pyrrole-protein adducts (PPAs), the PA exposure biomarker, were measured and found in 32% of surgically resected specimens from 34 patients with liver cancer in Hong Kong. Next, we delineated the mode of mutagenic and tumorigenic actions of retrorsine, a representative PA, in mice and human hepatocytes (HepaRG). Retrorsine induced DNA adduction, DNA damage, and activation of tumorigenic hepatic progenitor cells, which initiated hepatocarcinogenesis. PA mutational signature, as the unique molecular fingerprint of PA-induced mutation, was derived from exome mutations in retrorsine-exposed mice and HepaRG cells. Notably, PA mutational signature was validated in genomes of patients with PPA-positive liver cancer but not patients with PPA-negative liver cancer, confirming the specificity of this biomarker in revealing PA-associated liver cancers. Furthermore, we examined the established PA mutational signature in 1,513 liver cancer genomes and found that PA-associated liver cancers were potentially prevalent in Asia (Mainland China [48%], Hong Kong [44%], Japan [22%], South Korea [6%], Southeast Asia [25%]) but minor in Western countries (North America [3%] and Europe [5%]). CONCLUSIONS: This study provides a clinical indication of PA-associated liver cancer. We discovered an unexpectedly extensive implication of PA exposure in patients with liver cancer, laying the scientific basis for precautionary approaches and prevention of PA-associated human liver cancers.

Intestinal and hepatic biotransformation of pyrrolizidine alkaloid N-oxides to toxic pyrrolizidine alkaloids.

Pyrrolizidine alkaloids (PAs) are among the most significant groups of phytotoxins present in more than 6000 plants in the world. Hepatotoxic retronecine-type PAs and their corresponding N-oxides usually co-exist in plants. Although PA-induced hepatotoxicity is known for a long time and has been extensively studied, the toxicity of PA N-oxide is rarely investigated. Recently, we reported PA N-oxide-induced hepatotoxicity in humans and rodents and also suggested the association of such toxicity with metabolic conversion of PA N-oxides to the corresponding toxic PAs. However, the detailed biochemical mechanism of PA N-oxide-induced hepatotoxicity is largely unknown. The present study investigated biotransformation of four representative cyclic retronecine-type PA N-oxides to their corresponding PAs in both gastrointestinal tract and liver. The results demonstrated that biotransformation of PA N-oxides to PAs was mediated by both intestinal microbiota and hepatic cytochrome P450 monooxygenases (CYPs), in particular CYP1A2 and CYP2D6. Subsequently, the formed PAs were metabolically activated predominantly by hepatic CYPs to form reactive metabolites exerting hepatotoxicity. Our findings delineated, for the first time, that the metabolism-mediated mechanism of PA N-oxide intoxication involved metabolic reduction of PA N-oxides to their corresponding PAs in both intestine and liver followed by oxidative bioactivation of the resultant PAs in the liver to generate reactive metabolites which interact with cellular proteins leading to hepatotoxicity. In addition, our results raised a public concern and also encouraged further investigations on potentially remarkable variations in PA N-oxide-induced hepatotoxicity caused by significantly altered intestinal microbiota due to individual differences in diets, life styles, and medications.

First evidence of pyrrolizidine alkaloid N-oxide-induced hepatic sinusoidal obstruction syndrome in humans.

Pyrrolizidine alkaloids (PAs) are among the most potent phytotoxins widely distributed in plant species around the world. PA is one of the major causes responsible for the development of hepatic sinusoidal obstruction syndrome (HSOS) and exerts hepatotoxicity via metabolic activation to form the reactive metabolites, which bind with cellular proteins to generate pyrrole-protein adducts, leading to hepatotoxicity. PA N-oxides coexist with their corresponding PAs in plants with varied quantities, sometimes even higher than that of PAs, but the toxicity of PA N-oxides remains unclear. The current study unequivocally identified PA N-oxides as the sole or predominant form of PAs in 18 Gynura segetum herbal samples ingested by patients with liver damage. For the first time, PA N-oxides were recorded to induce HSOS in human. PA N-oxide-induced hepatotoxicity was further confirmed on mice orally dosed of herbal extract containing 170 µmol PA N-oxides/kg/day, with its hepatotoxicity similar to but potency much lower than the corresponding PAs. Furthermore, toxicokinetic study after a single oral dose of senecionine N-oxide (55 µmol/kg) on rats revealed the toxic mechanism that PA N-oxides induced hepatotoxicity via their biotransformation to the corresponding PAs followed by the metabolic activation to form pyrrole-protein adducts. The remarkable differences in toxicokinetic profiles of PAs and PA N-oxides were found and attributed to their significantly different hepatotoxic potency. The findings of PA N-oxide-induced hepatotoxicity in humans and rodents suggested that the contents of both PAs and PA N-oxides present in herbs and foods should be regulated and controlled in use.

Blood Pyrrole-Protein Adducts--A Biomarker of Pyrrolizidine Alkaloid-Induced Liver Injury in Humans.

Pyrrolizidine alkaloids (PAs) induce liver injury (PA-ILI) and is very likely to contribute significantly to drug-induced liver injury (DILI). In this study we used a newly developed ultra-high performance liquid chromatography-triple quadrupole-mass spectrometry (UHPLC-MS)-based method to detect and quantitate blood pyrrole-protein adducts in DILI patients. Among the 46 suspected DILI patients, 15 were identified as PA-ILI by the identification of PA-containing herbs exposed. Blood pyrrole-protein adducts were detected in all PA-ILI patients (100%). These results confirm that PA-ILI is one of the major causes of DILI and that blood pyrrole-protein adducts quantitated by the newly developed UHPLC-MS method can serve as a specific biomarker of PA-ILI.

Characteristic study of polycyclic aromatic hydrocarbons for fine and coarse particulates at Pastureland near Industrial Park sampling site of central Taiwan.

The concentrations of ambient air polycyclic aromatic hydrocarbons were measured in a farm area (Tunghai University Pastureland) between August 2001 and April 2002 in central Taiwan, Taichung. Particle-bound polycyclic aromatic hydrocarbons (PAHs) were collected on quartz filters, the collected sample was extracted with a dichloromethane (DCM)/n-hexane mixture (50/50, v/v) for 24 h, and then the extracts were subjected to gas chromatography-mass spectrometric analysis. The PM2.5 (fine particulate) and PM2.5-10 (coarse particulate) total PAHs concentrations at the Tunghai University Pastureland sampling site were found to be 180.62 ngm(-3) and 164.98 ngm(-3), respectively. In general, the concentrations of polycyclic aromatic hydrocarbons were higher in spring and winter than those of summer and autumn for either PM2.5 or PM2.5-10 in Pastureland in central Taiwan. Moreover, coarse particulates are the dominant species during the dust storm season (March and April) in central Taiwan.

Characterization, identification of ambient air and road dust polycyclic aromatic hydrocarbons in central Taiwan, Taichung.

The concentrations of ambient air polycyclic aromatic hydrocarbons (PAHs) were measured simultaneously in an industrial area (Taichung Industrial Park, TIP) and suburban area (Tunghai University, THU) in central Taiwan, Taichung. A total of samples were collected simultaneously at the two sites between August 2002 and March 2003. Particle-bound PAHs (p-PAHs) were collected on quartz filters and gas-phase PAHs (g-PAHs) on glass cartridges using polyurethane foam sampler, respectively. Both types of samples were extracted with dichloromethane/n-hexane mixture (50/50, v/v) for 24 h, then the extracts were subjected to gas chromatography/mass spectrometric analysis. Moreover, the roadside dust particle PAHs composition were also collected and analyzed at TIP, THU and traffic road sampling sites. The five main road lines in Taichung City were selected as traffic road sampling sites. Correlation studies between PAHs concentrations and meteorological parameters were revealed that temperature has greater effects (P>0.6) than other meteorological parameters such as wind speed, relative humidity and atmospheric pressure on g-PAHs and p-PAHs. PAHs sources were resolved by using principal component analysis and diagnostic ratios. The major sources of PAHs were combustion, traffic vehicle exhaust (diesel and gasoline engine), incinerator and industrial stationary sources at both sampling sites in central Taiwan.

Polycyclic aromatic hydrocarbons in the ambient air of suburban and industrial regions of central Taiwan.

The concentrations of gas-phase and particle-bound polycyclic aromatic hydrocarbons (PAHs) were measured simultaneously at an industrial area (Taichung Industrial Park) and a suburban area (Tunghai University Campus) in Taichung, Taiwan. Twenty-four hours samplings for two consecutive days were performed between August and December 2002 at both sampling sites. Ambient air particle-bound PAHs were collected on quartz filters and gas-phase PAHs were collected on glass cartridges using a PUF Sampler, respectively. Both types of samples were extracted with a DCM/n-hexane mixture (50/50, v/v) for 24 h, then the extracts were subjected to gas chromatography-mass spectrometric (GC-MS) analysis. Total PAHs concentrations at the Taichung Industrial Park (TIP) sampling site and the Tunghai University Campus (THUC) sampling site were found to be 1232.3+/-963.6 and 609.8+/-356.3 ng/m(3), respectively. Stationary combustion processes were mainly responsible for PAHs sources at the TIP sampling site, while traffic vehicle exhaust was the largest contributor for PAHs sources at the THUC sampling site.

Characterization of particulate, metallic elements of TSP, PM(2.5) and PM(2.5-10) aerosols at a farm sampling site in Taiwan, Taichung.

Atmospheric aerosol particles and metallic concentrations were monitored at the Experimental Farm of Tunghai University (EFTU) sampling site in this study. Total suspended particulate matter (TSP) was collected by using a PS-1 sampler at the farm-sampling site, in central Taiwan, from July 2001 to April 2002. At the same time, PM(2.5) and PM(2.5-10) were also measured with a Universal sampler from January 2002 to April 2002. Only subjects with the most complete data records on TSP sampling (N=43) and PM(10) sampling (N=23) were used in this analysis. Taichung Industrial Park, Taichung Kang Road (traffic) and a Hospital Incinerator surround the Experimental Farm of Tunghai University. Atmospheric concentrations of metallic elements were analyzed by a flame atomic absorption spectrophotometer (AA-680/G). The results indicated that the metallic elements Mg, Cu and Mn were the largest components in the TSP fraction; the metallic elements Fe and Cd were the largest composition in the PM(2.5-10) fraction; however, the metallic elements Pb, Zn, Cr and Ni were the largest abundance in the PM(2.5) fraction. The atmospheric metallic elements in the TSP, PM(2.5) and PM(2.5-10) fractions came different emission sources, such as soil, traffic, industry and resuspended particles.

Concentration of atmospheric particulates during a dust storm period in central Taiwan, Taichung.

In this study we monitored concentrations of particles in central Taiwan using PS-1 (GPS1 PUF Sampler) and Model 310 Universal Air Sampler (UAS) from 02/23/2001 to 03/12/2001 at two sampling sites. During this period, an Asian dust storm moved across central Taiwan from 3/3 to 3/6. The total ambient air particle concentrations during the dust storm period were than compared with previous data from this region. In general, the average total suspended particulate (TSP) concentration order was during dust storm period > after dust storm period > non-dust storm period at both HKITT (traffic) and THUC (rural) sampling sites. The ratio of PM2.5/PM10 was 60% before and after the dust storm period. However, this ratio was decreased to less than 50% during the dust storm. This demonstrates that the coarse particulate concentrations (PM2.5-10) increased during the dust storm period. In contrast the increase of ambient air particles concentrations after the Taiwan Chi-Chi Earthquake were mainly due to fine particles (PM2.5). And, the increased of ambient air particles concentrations after dust storm period were mainly coarse particle (PM2.5-10) concentrations in central Taiwan.

The measurements of ambient particulates (TSP, PM2.5, PM2.5-10), chemical component concentration variation, and mutagenicity study during 1998-2001 in central Taiwan.

During June 1998 and February 2001, the experiments of this study were conducted at four sampling sites (THUPB, THUC, HKIT and CCRT) with different characters (suburban, rural and traffic). The chemical components (Cl-, Na+, K+, Mg2+, Ca2+, Fe, Zn, Pb, Ni) in suspended particle were also analyzed simultaneously. The particulate mass concentrations are higher in the traffic site (CCRT) than the other sampling sites in this study. This is because that high traffic density flow characterized CCRT sampling site. Besides, the fine particle (PM2.5) concentration was the dominant species out of the total suspended particles in central Taiwan, Taichung. The same phenomenon is found in most of cities around the world. Moreover, chloride, nitrate sulfate and ammonium are higher in Taiwan than other sampling sites in the world. The results also indicated that the control of acidic and secondary aerosol pollutants have become an important issue in Taiwan. In addition, mutagenic assays on the organic extracts of airborne particulates at different sampling sites were also conducted in central Taiwan. The data obtained here also reflected that the mutagenicity of the suspended particulates are significantly higher in winter period than it occurred in summer period.

Emission characters of particulate concentrations and dry deposition studies for incense burning at a Taiwanese temple.

Suspended particulate concentrations were measured at the Tzu Yun Yen temple in the Taichung region of Taiwan. The temple performs traditional incense burning. A universal sampler and a micro-orifice uniform deposited impactor (MOUDI) sampler with a dry deposition plate were used to measure the particulate concentrations. The results show that the average PM2.5/PM10 ratio was 74% during the incense burning period at this temple. In addition, the average suspended particulate (PM10) element concentration of anthropogenic element Zn (495 ng/m3) was higher than the other anthropogenic elements (Pb, Mn, Ni, and Cd). Furthermore, the average mass size distribution was bimodal with major peaks occurring at 0.32-0.56 microm and 5.6-10 microm during the incense burning period. The dry deposition velocities of Cd used fine particulates (PM2.5) and suspended particulate (PM10) mode were 1.86 and 0.99 cm/s in this study, respectively.