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Perilla leaves are known to be a rich source of polyphenols, which have been shown to exhibit various biological effects. This study aimed to compare the bioefficacies and bioactivities of fresh (PLEf) and dry (PLEd) Thai perilla (Nga-mon) leaf extracts. Phytochemical analysis indicated that both PLEf and PLEd were abundant in rosmarinic acid and bioactive phenolic compounds. PLEd, which had higher levels of rosmarinic acid but lower levels of ferulic acid and luteolin than PLEf, exhibited greater effectiveness in a free radical scavenging assay. Furthermore, both extracts were found to suppress intracellular ROS generation and exhibit antimutagenic activity against food-borne carcinogens in S. typhimurium. They also attenuated lipopolysaccharide-induced inflammation in RAW 264.7 cells by inhibiting the expression of nitric oxide, iNOS, COX-2, TNF-α, IL-1β, and IL-6 through the suppression of NF-κB activation and translocation. However, PLEf exhibited a higher ability to suppress cellular ROS production and higher antimutagenic and anti-inflammatory activities than PLEd, which can be attributed to its combination of phytochemical components. Overall, PLEf and PLEd have the potential to serve as natural bioactive antioxidant, antimutagenic, and anti-inflammatory agents to achieve potential health benefits.

Background and purpose: Ulcerative colitis is a chronic inflammatory bowel disease that involves diffused inflammation of the large intestine. Omega-3 fatty acid (FA) has been known to regulate the inflammatory response associated with ulcerative colitis pathogenesis. Perilla frutescens is a valuable source of omega-3 FA and α-linolenic acid (ALA) contained in its seed oil. Therefore, the aim of this study was to evaluate the anti-inflammatory effect of Perilla seed oil (PSO) on colitis induced by dextran sulfate sodium (DSS) in a mouse model. Experimental approach: PSO was extracted using a cold-pressed extractor and FA composition of PSO was analyzed by GC-MS. Acute colitis in mice was induced with 3% DSS in drinking water for 7 days. Some mice were treated with PSO (20, 100, 200 mg/kg BW) for 3 weeks before the DSS administration. Sulfasalazine was used as a positive control. The clinical features, histopathologic, serum, and gene expression of proinflammatory cytokines in the colon were assessed. Finding/Results: PSO contained the highest proportion of ALA (61.51%). Furthermore, PSO pretreatment evidently reduced body weight loss, diminished diarrhea, gross bleeding, and DSS-induced colon shortening. PSO pretreatment attenuated histopathological changes in response to DSS-induced colitis. PSO pretreatment also markedly decreased inflammatory response in serum and the colon tissue of DSS-induced mice. Conclusion and implication: ALA in PSO is suggested to be mainly responsible for the reduction of DSS-induced colitis through suppressing inflammatory markers. PSO could be further developed as a functional health supplement, which would be beneficial for anti-inflammation in the colonic mucosa.

Osteoporosis is the result of an imbalance in the bone-remodeling process via an increase in osteoclastic activity and a decrease in osteoblastic activity. Our previous studies have shown that

Industrially, after the removal of oil from perilla seeds (PS) by screw-type compression, the large quantities of residual perilla seed meal (PSM) becomes non-valuable waste. Therefore, to increase the health value and price of PS and PSM, we focused on the biological effects of perilla seed oil (PSO) and rosmarinic acid-rich fraction (RA-RF) extracted from PSM for their role in preventing oxidative stress and inflammation caused by TNF-α exposure in an A549 lung adenocarcinoma culture model. The A549 cells were pretreated with PSO or RA-RF and followed by TNF-α treatment. We found that PSO and RA-RF were not toxic to TNF-α-induced A549 cells. Both extracts significantly decreased the generation of reactive oxygen species (ROS) in this cell line. The mRNA expression levels of IL-1β, IL-6, IL-8, TNF-α, and COX-2 were significantly decreased by the treatment of PSO and RA-RF. The Western blot indicated that the expression of MnSOD, FOXO1, and NF-κB and phosphorylation of JNK were also significantly diminished by PSO and RA-RF treatment. The results demonstrated that PSO and RA-RF act as antioxidants to scavenge TNF-α induced ROS levels, resulting in decreased the expression of MnSOD, FOXO1, NF-κB and JNK signaling pathway in a human lung cell culture exposed to TNF-α.

Thai perilla (Perilla frutescens) extracts, which contain a substantial quantity of bioactive substances including phenolics and flavonoids, have shown marked anti-inflammatory activities in several investigated models. In the present study, the effect of perilla seed extract (PSE) and seed meal extract (PSME) on TNF-α-induced inflammatory response in human lung adenocarcinoma A549 cells was investigated. The total phenolic and flavonoid contents in PSME was lower than PSE. Markedly, rosmarinic acid was identified as the main constituent in both extracts. However, the DPPH and ABTS assays indicated that the antioxidant capacity of PSME was equal to PSE. Moreover, the iron-binding activity of PSE and PSME were exhibited by complex formation with Fe3+-NTA, indicating that the extracts may inhibit hydroxyl radical production via Fenton reaction. In vitro cytotoxicity analysis showed that both PSE or PSME co-treated with TNF-α, at 24 h exposure, were not toxic to the A549 cells. Interestingly, PSE and PSME dramatically exhibited an anti-inflammatory activity by inhibiting the mRNA expression of pro-inflammatory cytokines, IL-1β, IL-6, IL-8, and TNF-α, but did not influence iNOS and COX-2 mRNA expressions. Moreover, both extracts significantly reduced reactive oxygen species (ROS) production in TNF-α-induced A549 cells. The findings presented in this paper suggest that PSE and PSME could mitigate TNF-α-mediated inflammatory responses via limiting pro-inflammatory cytokine expressions and decreasing ROS production. Thus, perilla seed and seed meal, the by-product of a perilla seed oil cold-pressed extraction process, could be developed as food supplements or functional foods for the prevention of inflammation-induced lung carcinogenesis development. Keywords: Human lung adenocarcinoma cell line, Inflammation, Perilla seed, Perilla seed meal, Tumor necrosis factor-alpha (TNF-α)

Particulate matter from forest fires (PMFF) is an environmental pollutant causing oxidative stress, inflammation, and cancer cell metastasis due to the presence of polycyclic aromatic hydrocarbons (PAHs). Perilla seed meal contains high levels of polyphenols, including rosmarinic acid (RA). The aim of this study is to determine the anti-oxidative stress, anti-inflammation, and anti-metastasis actions of rosmarinic acid rich fraction (RA-RF) from perilla seed meal and its underlying molecular mechanisms in A549 cells exposed to PMFF. PMFF samples were collected via the air sampler at the University of Phayao, Thailand, and their PAH content were analyzed using GC-MS. Fifteen PAH compounds were detected in PMFF. The PMFF significantly induced intracellular reactive oxygen species (ROS) production, the mRNA expression of pro-inflammatory cytokines, MMP-9 activity, invasion, migration, the overexpression of c-Jun and p-65-NF-κB, and Akt phosphorylation. Additionally, the RA-RF significantly reduced ROS production, IL-6, IL-8, TNF-α, and COX-2. RA-RF could also suppress MMP-9 activity, migration, invasion, and the phosphorylation activity of c-Jun, p-65-NF-κB, and Akt. Our findings revealed that RA-RF has antioxidant, anti-inflammatory, and anti-metastasis properties via c-Jun, p-65-NF-κB, and Akt signaling pathways. RA-RF may be further developed as an inhalation agent for the prevention of lung inflammation and cancer metastasis induced by PM exposure.

Perilla is a rich source of polyphenols, which exhibits antioxidant, anti-inflammatory activities, and a variety of biological effects. The effect of differential solvents on the polyphenols, flavonoids, rosmarinic acid (RA), antiinflammatory and antioxidant activities of perilla leaf require investigation. In this study, perilla leaf was extracted with 70% ethanol and sequentially fractionated according to the solvent’s polarity with hexane, dichloromethane, ethyl acetate, and water. Samples were subjected to the bioactive compound measurements. The antioxidant and antiinflammation nature of perilla was analyzed based on the scavenging effects on DPPH•, ABTS•+, O2•- and nitric oxide (NO), as well as FRAP assay, and determination of the inhibition effects on NO, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) production in the cell-based study. The results indicate that among all different solvents used for sequential fractionation, ethyl acetate (EtOAc) was most effective in the separation of anti-oxidative and antiinflammatory compounds in the perilla leaf extract. These properties can partly be due to the presence of polyphenols, flavonoids, and also RA. It can be demonstrated here that, the perilla leaf EtOAc fraction could be used as a natural active pharmaceutical ingredient for dietary supplements and nutraceuticals.

Background and purpose: Ulcerative colitis is a chronic inflammatory bowel disease that involves diffused inflammation of the large intestine. Omega-3 fatty acid (FA) has been known to regulate the inflammatory response associated with ulcerative colitis pathogenesis. Perilla frutescens is a valuable source of omega-3 FA and α-linolenic acid (ALA) contained in its seed oil. Therefore, the aim of this study was to evaluate the anti-inflammatory effect of Perilla seed oil (PSO) on colitis induced by dextran sulfate sodium (DSS) in a mouse model. Experimental approach: PSO was extracted using a cold-pressed extractor and FA composition of PSO was analyzed by GC-MS. Acute colitis in mice was induced with 3% DSS in drinking water for 7 days. Some mice were treated with PSO (20, 100, 200 mg/kg BW) for 3 weeks before the DSS administration. Sulfasalazine was used as a positive control. The clinical features, histopathologic, serum, and gene expression of proinflammatory cytokines in the colon were assessed. Finding/Results: PSO contained the highest proportion of ALA (61.51%). Furthermore, PSO pretreatment evidently reduced body weight loss, diminished diarrhea, gross bleeding, and DSS-induced colon shortening. PSO pretreatment attenuated histopathological changes in response to DSS-induced colitis. PSO pretreatment also markedly decreased inflammatory response in serum and the colon tissue of DSS-induced mice. Conclusion and implication: ALA in PSO is suggested to be mainly responsible for the reduction of DSS-induced colitis through suppressing inflammatory markers. PSO could be further developed as a functional health supplement, which would be beneficial for anti-inflammation in the colonic mucosa.

For centuries, many kinds of native plants and their products have been used for the treatment of gastric ulcers by traditional healers in Phayao province. The current study aimed to investigate the polyphenol content in some of these medicinal plants and to point out the relationship between their antioxidant capacity and anti-inflammatory activities. Six species were selected based on ethnopharmacologic considerations: Punica granatum L., Psidium guajava L., Careya arborea Roxb., Gochnatia decora (Kurz) Cabr., Shorea obtusa Wall. ex Blume, and Ficus hispida L.f. The leaves or bark of these plants were extracted with 70% ethanol and water. Anti-inflammatory and antioxidant activities of the extracts were analyzed based on nitric oxide (NO) and proinflammatory cytokine production in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages and through the determination of scavenging activity. The results demonstrated that the ethanol extract from P. granatum and P. guajava leaves significantly inhibited NO production by suppressing nitric oxide synthase. The extracts also inhibited tumor necrosis factor-&alpha, interleukin-1, and interleukin-6 in terms of both mRNA and protein levels and possessed high antioxidants. These extracts were shown to contain the highest amount of polyphenols. Our study concluded that among the plants studied, P. granatum and P. guajava have the most significant anti-inflammatory and antioxidant activities and polyphenols. These plants may have the potential for use in gastric ulcer therapy due to their indicated properties. Future research should focus on the isolation of their active compounds and their in vivo biological activities. Their beneficial applications need to be warranted by such evidence.

Functional foods (FF) are commonly consumed by Asians, and this trend has increased in recent years. Despite the reported health benefits of FF, it is necessary scrutiny and updates of the underpinning research are important. The first international conference on functional food innovation in Asia (IFFA 2018) took place on January 22nd- 24th, 2018, at the University of Phayao, Thailand. Domestic and international speakers, researchers, nutritionists, dieticians, research scholars and students shared their knowledge and experience in FF research. Key features were the potential beneficial roles of FF in health and disease, the current situation with FF in Asia and innovative trends. The IFFA 2018 involved 2 keynote speakers, 34 invited speakers and 10 sessions. About 250 people from across Asia participated. Key themes, discussions, innovative opportunities, and future directions to link research in academia with health-directed applications as FF are summarised.

Gastric ulcers are a common problem in upper gastrointestinal tract (GI) disorders. Nonsteroidal anti-inflammatory drugs (NSAIDs) are one of the most aggressive factors leading to inducing gastric ulcers. Natural products with lower toxicity and safety are currently sought as a potential source to minimize the effect of the gastric ulcers.Perilla frutescensor Nga-mon (in Thai) leaves are rich in rosmarinic acid (RA), which has antioxidant, anti-inflammatory, and anticancer effects. This study investigates the protective effect of ethanolic extract (EE) and aqueous fraction (AF) fromPerilla frutescensleaves, which are rich in RA, on indomethacin- (IND-) induced gastric ulcer in a rat model. The EE at the doses of 50 and 500 mg/kg body weight, AF at the doses of 50, 250, and 500 mg/kg body weight, or famotidine (a standard drug) were administered for 14 days prior to ulcer induction. The ulceration was performed by intragastric administration of IND. Gross gastric ulcers and biological and histological parameters were examined. The pretreatment with AF had more significant effects than EE, including reduced ulcer index, decreased gastric secretion volume and decreased acidity, but it had an elevated gastric pH relative to the IND-induced gastric ulcer. In a histopathological study, the EE and AF decreased mucosal ulcer, inflammatory infiltration, and degenerative lining cells. The IND-induced expression of inflammatory mediators was significantly attenuated with EE and AF. The experiment also remarkably showed the preservation of mucus and apoptosis protection of EE and AF on a gastric mucosal ulcer. The findings demonstrated that the EE and AF of perilla leaves were capable of protecting the stomach against gastric ulcers induced by IND through anti-inflammatory and antiapoptotic mechanisms that should be further investigated. It is suggested thatPerilla frutescensleaf could be a potential alternative source of RA as a therapeutic agent and food supplement for NSAID-induced gastric injuries.

Perilla frutescens is commonly used as an n-3-fatty acid source for people living in Northern Thailand. However, cultivated areas are limited because it apparently requires specific growth conditions. Our results suggested that perilla seeds grown in higher altitude in Maehongson province contained more ALA contents than seeds collected from Chiangrai and Nan province. Furthermore, mass spectrometry (MS) proteomic and gene expression analysis revealed that the increase of KASII and FAD3 proteins associated with the mRNA expression and the ALA production. Predictive bioinformatics analysis demonstrated two important transcription factor binding sites, AP2 and B3, which responsible for KASII and FAD3 genes, respectively. The AP2 and B3 transcription factor families were known to be responsible for abiotic stress such as drought and temperature changes. In conclusion, high production of ALA by perilla apparently relies on the altitudes. Moreover, plant responses to abiotic stress environments contribute to the increase of KASII and FAD3 gene and protein expression. However, responsive transcription factors will be further studied for a proof of concept and additionally improving the growing methods in order to increase perilla productivity.

The ethanolic extracts of leaves of Psidium guajava (PG), Pandanous odorus (PO),Rhinacanthus nasutus (RN) contained polyphenolic compounds in the order of PG > RN > PO. All the extracts exhibited 2,2-diphenyl-1-picryhydrazyl (DPPH) and 2,2’-azino-bis 3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radicals scavenging activities. PG showed a marked antioxidant effect whereas PO and RN were comparable. Interestingly, they were able to bind with ferric (Fe3+) and ferrous (Fe2+) ion and completed within 5 min of incubation times. In accordance with previous results, PG extract could effectively protect erythrocytes from iron induced hemolysis with the activity higher than PO and RN. Based on all results, tested leaf extracts of three Thai medicinal plants might be used for alleviating or preventing harmfulness in many oxidative stress disorders. Key words: Psidium guajava, Pandanous odorus, Rhinacanthus nasutus, antioxidant, iron binding capacity, anti-hemolysis activity.

A checklist covering a total of 67 Recent non-marine ostracod species from Thailand, belonging to 33 genera and seven families (Cyprididae, Candonidae, Ilyocyprididae, Notodromadidae, Darwinulidae, Limnocytheridae, Loxoconchidae), is presented. Twenty-five of these are considered endemic to the Oriental region, 17 of which are Thai endemics. Two species (Hemicypris exiqua Broodbakker, 1983, and Notodromas sinensis NealZhao, 1991) are new records for the Oriental region, and four species (Cypretta seurati Gauthier, 1929, Chrissia ceylonica (Daday, 1898), Chrissia formosa (Klie, 1938), and Dolerocypris fasciata (O.F. Müller, 1776)) are new record for Thailand. The most diverse subfamily in Thailand is the Cypricercinae, with 22 species. The most diverse genera are Strandesia Stuhlmann, 1888 (eight species), followed by Pseudostrandesia SavatenalintonMartens, 2009 (seven species) and Stenocypris Sars, 1889 (five species). Other enigmatic groups and new taxa occur in the checklist, but these need to be described elsewhere in the future. For example, several species of Cypridopsinae Kaufmann, 1900, Cyclocypridinae Kaufmann, 1900, Candoninae Kaufmann, 1900 and other groups await description. Considering regional distributions based on frequency of occurrences, rare species constitute a large fraction, whereas very common species are minor elements. Most of the records were reported from the Northern and Northeastern parts of Thailand. Sampled habitat types ranged from ponds, (oxbow) lakes, swamps, reservoirs, springs, canals, rivers, rice fields, waterfalls, and damp leaf litter. Most of the Thai ostracod fauna exists in a wide range of habitats. A brief discussion on the distribution of described species is included with the list. Additionally, in the present contribution, we describe two new species, Dolerocypris sisaketensis n. sp. and Hungarocypris suranareeae n. sp., from the Northeastern part of Thailand. The main character of Dolerocypris sisaketensis n. sp. is the presence of a spine on the postero-ventral part of the right valve; this feature sets it apart from other Dolerocypris species. Hungarocypris suranareeae n. sp. is the second species of this genus in Thailand and the third one in Southeast Asia. It is clearly distinguished from other Hungarocypris species by the serration on both valves. The genera Dolerocypris and Hungarocypris are briefly discussed and a key to the species of Hungarocypris is provided.

Naresuan Phayao Journal, 9, 2

There has been increasing interest in finding natural antioxidants to prevent free radical damage and retard the progress of chronic inflammatory diseases. Our previous data demonstrated the strong antioxidant properties of polyphenolics in Mamao seed (MS) and Mamao marc (MM) extracts. In this study we further investigated the effect of MS and MM polyphenolics on hydrogen peroxide (H 2 O 2 )-induced apoptosis and tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation, using human breast epithelial (MCF10A) cells. MS and MM extracts conferred dose-dependent protection against H 2 O 2 -induced apoptosis by inhibiting PARP/caspase-3 cleavage, inducing anti-apoptotic Bcl-2 expression, and down-regulating pro-apoptotic Bax. Moreover, MS and MM polyphenolics inhibited TPA-induced COX-2 and NF-κB activation by blocking the degradation of cytoplasmic IκBα, as well as subsequent nuclear translocation of p65 and attenuation of the activation of ERK, but not JNK and p38. These data establish the molecular mechanism for the anti-apoptotic and anti-inflammatory effects of MS and MM polyphenolics.

Purslane weed has long been used for animal feed in Thailand but has been underutilized, and no published data exist on the chemical and nutritional qualities of Thai wild purslane. In this study, we aimed to determine the microchemical components of purslane stem, leaf, and flower, including the phenolic acid, flavonoid, ascorbic acid, β-carotene, and fatty acid content. The water extract of the flower fraction contained the highest total phenolic acid content and had the highest O2-scavenging activities, whereas leaf contained the highest amount of total flavonoids and ascorbic acid. The β-carotene content was not significantly different in leaf and flower fractions but was significantly greater than that in stem (P < 0.05). The predominant phenolic acid was chlorogenic acid for all fractions. Rutin was the major flavonoid found in leaf, and myricetin was highest in flower and stem. Alpha linolenic acid (18:3n-3) content ranged from 16% (149 mg per 100-g sample) of total fatty acid in stem to 50% (523 mg per 100-g sample) in leaf. We suggest that Thai wild purslane could be considered a nutritional source for animal feed or an excellent vegetable in the human diet.

The objective of this study was to investigate the in vitro bioactivities associated with the content of phytochemicals in the extracts from perilla seed meal extract (PSME) compared with dietary seed (PSE). PSE had higher total phenolics and flavonoids content than PSME. However, hydrophilic phytochemical contents in PSME were quantitatively equivalent to PSE. Rosmarinic acid was predominantly found in both extracts. Cell viability and anti‐mutagenicity testing demonstrated that PSE and PSME were biosecured and non‐genotoxic. Both extracts strongly scavenged free radicals and significantly reduced reactive oxygen species (ROS) production. The extracts drastically diminished nitric oxide (NO) production of LPS‐treated RAW 264.7 cells via iNOS mRNA expression. The expression of IL‐6 and COX‐2 were evidently inhibited by these extracts. It could be concluded that PSE and PSME clearly showed in vitro anti‐mutagenicity, antioxidant and anti‐inflammatory capacities. In particular, the by‐product perilla seed meal could be considered as a high nutritive functional food. PRACTICAL APPLICATIONS: This study suggests that the seed meals, a by‐product from seed oil industry, can be utilized as a valuable dietary source for humans and animals. The high content of polyphenols and their bioactivities can be developed as functional foods, and excipients and fillers in pharmaceuticals and nutraceuticals production. Moreover, recycling of the by‐product seed meals should also reduce environmental and sanitary pollution.

Ethanolic extracts of 30 Thai medicinal plants, traditionally used as alternative treatments in diabetes, were evaluated for antioxidative activity by the 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) method. They were evaluated in vitro for oxidative stress by thiobarbituric acid-reactive substance (TBARS) assay in pooled plasma of diabetic patients compared to without treatment of the extracts (control). The extracts were also assayed for protein glycation. The results showed that five plants had strong antioxidant activity: Phyllanthus emblica Linn. (PE), Terminalia chebula Retz. (TC), Morinda citrifolia Linn. (MC), Kaempferia parviflora Wall. (KP) and Houttuynia cordata Thunb.(HC), respectively. Thirty plant extracts were good correlation between total antioxidant activity and antiradical activity by TBARS as well as by glycation (r = 0.856, p

Ethanolic extracts from various parts of 26 Thai indigenous plants were examined for phenolic constituents and free radical scavenging capacity, to determine their potential as a source of natural antioxidants. Total phenolic content and total flavonoid content were evaluated according to the Folin-Ciocalteu procedure, and a colorimetric method, respectively. The results showed that total phenolic compounds and flavonoid content were higher in seed extracts of berries used in wine production, while the levels in extracts obtained from herbs and vegetables were lower. Chewing plants which have an astringent taste gave a significantly higher total phenolic content and flavonoid content. Antiradical activity determined from 1/EC50 by the DPPH radical-scavenging method was highest in wine production seeds and chewing plants. The correlation coefficient from regression analysis showed a positive relationship between total phenolic and total flavonoid content (r = 0.9). The results suggest that ethanolic extracts of some Thai indigenous plants exhibit a potential for use as natural antioxidants.

Effects of extracts of a plant, which has been used as a traditional medicine for treating diabetes on glucose transport activity was evaluated in cultured L8 muscle cells. The aqueous extract of Canna indica root (CI) at doses of 0.1-0.5 mg/ml, which contains total phenolic compounds equivalent to 6-30 microg of catechin caused a dose- and time-dependent induction of 2-deoxy-[3H]glucose (2-DG) uptake activity. The induced 2-DG uptake was significantly increased within 8 h and reached a maximum by 16 h. The CI extract increased the amount of glucose transporter isoforms 1 (GLUT1) and 4 (GLUT4) at the cell surface and enhanced expression of GLUT1 protein. Cycloheximide treatment almost completely reversed CI-induced 2-DG uptake to the basal level. Exposure of muscle cells to wortmannin and SB203580 diminished CI-mediated glucose uptake by 38 and 14%, respectively. The effect of CI and insulin was partially additive. Phytochemical analysis detected the presence of flavonoids and catechol in the CI. Taken together, these data provide evidence for differential effects of CI on regulated-glucose transport in muscle cells. Our findings suggest that GLUT1 protein synthesis and the activation of phosphatidylinositol 3-kinase (PI3K) are critical for the increase in glucose transporter activity at the plasma membrane and essential for the maximal induction of glucose transport by CI in L8 muscle cells.

Lung cancer incidence among Northern Thai women is one of the highest in Asia (an annual age-adjusted incidence rate of 37.4 per 100,000), and the incidence rate significantly differs by geographical districts. Therefore, we conducted a comparative study of women living in the Sarapee area, which showed the highest (crude incidence rate, 40.9), and the Chom Tong area, which had one of the lowest incidence rates (8.5) in Chiang Mai Province, despite the two areas' geographical and cultural closeness. The women in this study were either family members of lung cancer patients or their neighbors. To find clues to the etiology of lung cancer, this study used various epidemiological and biochemical approaches: interviewing on lifestyle factors, duplicate meals, chemical examination of drinking water, biochemical analysis of sera, mutagenicity test of urine, and monitoring of fungi and bacteria in the living environment. We found that tobacco smoking (Khiyo, local cigars) was less frequently observed in Sarapee (high incidence), compared with Chom Tong (low incidence), and that the history of chronic benign respiratory diseases was the most distinct event among women in Sarapee, resulting in a significantly increased percentage of those with a history of both benign respiratory diseases and tobacco smoking. This population revealed increased levels of serum tumor necrosis factor (TNF)-alpha, an endogenous tumor promoter. Furthermore, significantly increased urine mutagenicity was found to be closely associated with history of benign respiratory disease in Sarapee. The fungus which was most commonly found in the air inside houses in Sarapee was identified as Microsporum canis. Additionally, significantly increased serum concentrations of a constituent of the fungus were found in Sarapee women, compared with those in Chom Tong. Our results suggest that tobacco (Khiyo) smoking alone may not be able to explain the very high incidence of female lung cancer in Northern Thailand, and that chronic benign respiratory disease, possibly caused by the infection of fungi such as M. canis, is likely to be involved in the etiology of female lung cancer in North Thailand.