Repurposing Salicylamides to Combat Phytopathogenic Bacteria and Induce Plant Defense Responses.

Based on the research strategy of "drug repurposing", a series of derivatives and marketed drugs that containing salicylic acid skeleton were tested for their antibacterial activities against phytopathogens. Salicylic acid can not only regulate some important growth metabolism of plants, but also induce plant disease resistance. The bioassay results showed that the salicylamides exhibited excellent antibacterial activity. Especially, oxyclozanide showed the best antibacterial effect against Xanthomonas oryzae, Xanthomonas axonopodis pv. citri and Pectobacterium atroseptica with MICs of 0.78, 3.12 and 12.5 µg.mL-1, respectively. In vivo experiments with rice bacterial leaf blight had further demonstrated that oxyclozanide exhibited stronger antibacterial activity than the commercial bactericide, thiodiazole copper. Oxyclozanide could induce plant defense responses through the determination of salicylic acid content and the activities of defense-related enzymes including CAT, POD, and SOD in rice. The preliminarily antibacterial mechanism study indicated that oxyclozanide exhibited the antibacterial activity by disrupting cell integrity and reducing bacterial pathogenicity. Additionally, oxyclozanide could induce plant defense responses through the determination of salicylic acid content.

Drug repurposing strategy part 1: from approved drugs to agri-bactericides leads.

Phytopathogenic bacteria are a major cause of crop mortality and yield reduction, especially in field cultivation. The lack of effective chemistry agri-bactericides is responsible for challenging field prevention and treatment, prompting the development of long-lasting solutions to prevent, reduce, or manage some of the most devastating plant diseases facing modern agriculture today and in the future. Therefore, there is an urgent need to find lead drugs preventing and treating phytopathogenic bacterial infection. Drug repurposing, a strategy used to identify novel uses for existing approved drugs outside of their original indication, takes less time and investment than Traditional R&D Strategies in the process of drug development. Based on this method, we conduct a screen of 700 chemically diverse and potentially safe drugs against Xanthomonas oryzae PV. oryzae ACCC 11602 (Xoo), Xanthomonas axonopodis PV. citri (Xac), and Pectobacterium atrosepticum ACCC 19901 (Pa). Furthermore, the structure-activity relationship and structural similarity analysis of active drugs classify potent agri-bactericides into 8 lead series: salicylanilides, cationic nitrogen-containing drugs, azole antifungals, N-containing group, hydroxyquinolines, piperazine, kinase inhibitor and miscellaneous groups. MIC values were evaluated as antibacterial activities in this study. Identifying highly active lead compounds from the screening of approved drugs and comparison with the currently applied plant pathogenic bactericide to validate the bactericidal activity of the best candidates and assess if selected molecules or scaffolds lead to develop new antibacterial agents in the future. In conclusion, this study provides a possibility for the development of potent and highly selective agri-bactericides leads.

Antibacterial activity and action mechanism of flavonoids against phytopathogenic bacteria.

As the most difficult to control in plant disease, phytopathogenic bacteria cause huge losses to agricultural products and economy worldwide. However, the commercially available bactericides are few and enhance pathogen resistance. To alleviate this situation, 50 flavonoids were evaluated for their antibacterial activities and mechanism of action against two intractable plant bacterial pathogens. The results of bioassays showed that most of the flavonoids exhibited moderate inhibitory effects against Xanthomonas oryzae (Xo) and Xanthomonas axonopodis pv citri (Xac). Remarkably, kaempferol showed excellent antibacterial activity against Xo in vitro (EC50 = 15.91 µg/mL) and quercetin showed the best antibacterial activity against Xac in vitro (EC50 = 14.83 µg/mL), which was better than thiodiazole copper (EC50 values against Xo and Xac were 16.79 µg/mL, 59.13 µg/mL, respectively). Subsequently, in vivo antibacterial activity assay further demonstrated kaempferol exhibited a stronger control effect on bacterial infections than thiodiazole copper. Then, the preliminary antibacterial mechanism of kaempferol was investigated by ultrastructural observations, transcriptomic, qRT-PCR analysis and biochemical index determination. These results showed that kaempferol mainly exerted bacteriostatic effects at the molecular level by affecting bacterial energy metabolism, reducing pathogenicity, and leading to disruption of cellular integrity, leakage of contents and cell death eventually.

Design, Synthesis, and Antimicrobial Activity of Quindoline Derivatives Inspired by the Cryptolepine Alkaloid.

Based on the structural characteristics of the cryptolepine alkaloid, a series of new quindoline derivatives bearing various substituents were prepared and evaluated for their fungicidal and antibacterial activities. Bioassay results showed that compound D7 displayed superior in vitro fungicidal activities against Sclerotinia sclerotiorum, Botrytis cinerea, Fusarium graminearum, and Rhizoctonia solani with EC50 values of 0.780, 3.62, 1.59, and 2.85 µg/mL, respectively. Compound A7 showed apparent antibacterial activities toward Xanthomonas oryzae pv. oryzae with a minimum inhibitory concentration (MIC) value of 3.12 µg/mL. Significantly, in vivo antifungal activity suggested that the curative effect (98.3%) of compound D7 was comparable to that of the positive control azoxystrobin (96.7%) at 100 µg/mL. Preliminary mechanistic studies showed that compound D7 might cause mycelial abnormality of S. sclerotiorum, cell membrane breakage, accumulation of reactive oxygen species (ROS), and inhibition of sclerotia formation. Therefore, compound D7 could be a novel broad-spectrum fungicidal candidate against plant fungal diseases.

Design, synthesis and biological evaluation of novel evodiamine and rutaecarpine derivatives against phytopathogenic fungi.

Evodiamine and rutaecarpine are two alkaloids isolated from traditional Chinese herbal medicine Evodia rutaecarpa, which have been reported to have various biological activities in past decades. To explore the potential applications for evodiamine and rutaecarpine alkaloids and their derivatives, various kinds of evodiamine and rutaecarpine derivatives were designed and synthesized. Their antifungal profile against six phytopathogenic fungi Rhizoctonia solani, Botrytis cinerea, Fusarium graminearum, Fusarium oxysporum, Sclerotinia sclerotiorum, and Magnaporthe oryzae were evaluated for the first time. Furthermore, a series of modified imidazole derivatives of rutaecarpine were synthesized to investigate the structure-activity relationship. The results of antifungal activities in vitro showed that imidazole derivative of rutaecarpine A1 exhibited broad-spectrum inhibitory activities against R. solani, B. cinerea, F. oxysporum, S. sclerotiorum, M. oryzae and F. graminearum with EC50 values of 1.97, 5.97, 12.72, 2.87 and 16.58 µg/mL, respectively. Preliminary mechanistic studies showed that compound A1 might cause mycelial abnormalities of S. sclerotiorum, mitochondrial distortion and swelling, and inhibition of sclerotia formation and germination. Moreover, the curative effects of compound A1 were 94.7%, 81.5%, 80.8%, 65.0% at 400, 200, 100, 50 µg/mL in vivo experiments, which was far more effective than the positive control azoxystrobin. Significantly, no phytotoxicity of compound A1 on oilseed rape leaves was observed obviously even at a high concentration of 400 µg/mL. Therefore, compound A1 is expected to be a novel leading structure for the development of new antifungal agents.

Design, Synthesis, and Antifungal Evaluation of Luotonin A Derivatives against Phytopathogenic Fungi.

Crop diseases caused by fungi threaten food security and exacerbate the food crisis. Inspired by the application of fungicide candidates from natural products in agrochemical discovery, a series of luotonin A derivatives were designed, synthesized, and evaluated for their antifungal activities against five plant fungi. Most of these compounds exhibited significant fungicidal activity against Botrytis cinerea in vitro with EC50 values less than 1 µg/mL. Among them, compounds w7, w8, w12, and w15 showed superior antifungal activity against B. cinerea with EC50 values of 0.036, 0.050, 0.042, and 0.048 µg/mL, respectively, which were more potent than boscalid (EC50 = 1.790 µg/mL). Preliminary mechanism studies revealed that compound w7 might pursue its antifungal activity by disrupting the fungal cell membrane and cell wall. Moreover, in vivo bioassay also indicated that compound w7 could be effective for the control of B. cinerea. The above results evidenced the potential of luotonin A derivatives as novel and promising candidate fungicides.

Antifungal Activity and Action Mechanism Study of Coumarins from Cnidium monnieri Fruit and Structurally Related Compounds.

The increasing resistance of plant diseases caused by phytopathogenic fungi highlights the need for highly effective and environmentally benign agents. The antifungal activities of Cnidium monnieri fruit extracts and five isolated compounds as well as structurally related coumarins against five plant pathogenic fungi were evaluated. The acetone extract, which contained the highest amount of five coumarins, showed strongest antifungal activity. Among the coumarin compounds, we found that 4-methoxycoumarin exhibited stronger and broader antifungal activity against five phytopathogenic fungi, and was more potent than osthol. Especially, it could significantly inhibit the growth of Rhizoctonia solani mycelium with an EC50 value of 21 µg mL-1 . Further studies showed that 4-methoxycoumarin affected the structure and function of peroxisomes, inhibited the ß-oxidation of fatty acids, decreased the production of ATP and acetyl coenzyme A, and then accumulated ROS by damaging MMP and the mitochondrial function to cause the cell death of R. solani mycelia. 4-Methoxycoumarin presented antifungal efficacy in a concentration- dependent manner in vivo and could be used to prevent the potato black scurf. This study laid the foundation for the future development of 4-methoxycournamin as an alternative and friendly biofungicide.

Antifungal Exploration of Quinoline Derivatives against Phytopathogenic Fungi Inspired by Quinine Alkaloids.

Enlightened from our previous work of structural simplification of quinine and innovative application of natural products against phytopathogenic fungi, lead structure 2,8-bis(trifluoromethyl)-4-quinolinol (3) was selected to be a candidate and its diversified design, synthesis, and antifungal evaluation were carried out. All of the synthesized compounds Aa1-Db1 were evaluated for their antifungal activity against four agriculturally important fungi, Botrytis cinerea, Fusarium graminearum, Rhizoctonia solani, and Sclerotinia sclerotiorum. Results showed that compounds Ac3, Ac4, Ac7, Ac9, Ac12, Bb1, Bb10, Bb11, Bb13, Cb1. and Cb3 exhibited a good antifungal effect, especially Ac12 had the most potent activity with EC50 values of 0.52 and 0.50 µg/mL against S. sclerotiorum and B. cinerea, respectively, which were more potent than those of the lead compound 3 (1.72 and 1.89 µg/mL) and commercial fungicides azoxystrobin (both >30 µg/mL) and 8-hydroxyquinoline (2.12 and 5.28 µg/mL). Moreover, compound Ac12 displayed excellent in vivo antifungal activity, which was comparable in activity to the commercial fungicide boscalid. The preliminary mechanism revealed that compound Ac12 might cause an abnormal morphology of cell membranes, an increase in membrane permeability, and release of cellular contents. These results indicated that compound Ac12 displayed superior in vitro and in vivo fungicidal activities and could be a potential fungicidal candidate against plant fungal diseases.

Design, Synthesis, and Structure-Activity Relationship Studies of Magnolol Derivatives as Antifungal Agents.

Plant pathogenic fungi seriously affect agricultural production and are difficult to control. The discovery of new leads based on natural products is an important way to innovate fungicides. In this study, 30 natural-product-based magnolol derivatives were synthesized and characterized on the basis of NMR and mass spectroscopy. Bioactivity tests on phytopathogenic fungi (Rhizoctonia solani, Fusarium graminearum, Botrytis cinerea, and Sclerotinia sclerotiorum) in vitro of these compounds were performed systematically. The results showed that 11 compounds were active against four kinds of phytopathogenic fungi with EC50 values in the range of 1.40-20.00 µg/mL, especially compound L5 that exhibited excellent antifungal properties against B. cinerea with an EC50 value of 2.86 µg/mL, approximately 2.8-fold more potent than magnolol (EC50 = 8.13 µg/mL). Moreover, compound L6 showed the highest antifungal activity against F. graminearum and Rhophitulus solani with EC50 values of 4.39 and 1.40 µg/mL, respectively, and compound L7 showed good antifungal activity against S. sclerotiorum. Then, an in vivo experiment of compound L5 against B. cinerea was further investigated in vivo using infected tomatoes (curative effect, 50/200 and 36%/100 µg/mL). The physiological and biochemical studies illustrated that the primary action mechanism of compound L5 on B. cinerea might change the mycelium morphology, increase cell membrane permeability, and destroy the function of mitochondria. Furthermore, structure-activity relationship (SAR) studies revealed that hydroxyl groups play a key role in antifungal activity. To sum up, this study provides a reference for understanding the application of magnolol-based antifungal agents in crop protection.

Design and Discovery of Novel Antifungal Quinoline Derivatives with Acylhydrazide as a Promising Pharmacophore.

Inspired by natural 2-quinolinecarboxylic acid derivatives, a series of quinoline compounds containing acylhydrazine, acylhydrazone, sulfonylhydrazine, oxadiazole, thiadiazole, or triazole moieties were synthesized and evaluated for their fungicidal activity. Most of these compounds exhibited excellent fungicidal activity in vitro. Significantly, compound 2e displayed the superior in vitro antifungal activity against Sclerotinia sclerotiorum, Rhizoctonia solani, Botrytis cinerea, and Fusarium graminearum with the EC50 values of 0.39, 0.46, 0.19, and 0.18 µg/mL, respectively, and were more potent than those of carbendazim (EC50, 0.68, 0.14, >100, and 0.65 µg/mL, respectively). Moreover, compound 2e could inhibit spore germination of F. graminearum. Preliminary mechanistic studies showed that compound 2e could cause abnormal morphology of cell walls and vacuoles, loss of mitochondrion, increases in membrane permeability, and release of cellular contents. These results indicate that compound 2e displayed superior fungicidal activities and could be a potential fungicidal candidate against plant fungal diseases.

Integrated Proteomics and Transcriptomics Analyses Reveals the Possible Antifungal Mechanism of an Indoloquinoline Alkaloid Neocryptolepine against Rhizoctonia solani.

Rhizoctonia solani causes serious plant diseases. Neocryptolepine presented the significant antifungal activity against R. solani, however the mode of action is unclear. In this paper, we investigated the potential mode of action of neocryptolepine against R. solani integrated the proteomics and transcriptomics. Results showed that after treatment with neocryptolepine, 1012 differentially expressed proteins and 10 920 differentially expressed genes of R. solani were found, most of them were enriched in mitochondrial respiratory chain. It affected oxidative phosphorylation led to the enrichment of ROS and the decrease of MMP, and inhibited complex III activity with the inhibition rate of 63.51% at 10 µg/mL. The mitochondrial structural and function were damaged. Cytochrome b-c1 complex subunit Rieske (UQCRFS1) with the high binding score to neocryptolepine was found as a potential target. In addition, it inhibited the sclerotia formation and presented antifungal efficacy by decreasing the diameter of a wound in potato in a concentration-dependent manner. Above results indicated that neocryptolepine inhibited the complex III activity by binding UQCRFS1 and blocked the ion transfer to cause the death of R. solani mycelia. This study laid the foundation for the future development of neocryptolepine as an alternative biofungicide.

Design, Synthesis, and Antifungal Evaluation of Cryptolepine Derivatives against Phytopathogenic Fungi.

Inspired by the widely antiphytopathogenic application of diversified derivatives from natural sources, cryptolepine and its derivatives were subsequently designed, synthesized, and evaluated for their antifungal activities against four agriculturally important fungi Rhizoctonia solani, Botrytis cinerea, Fusarium graminearum, and Sclerotinia sclerotiorum. The results obtained from in vitro assay indicated that compounds a1-a24 showed great fungicidal property against B. cinerea (EC50 < 4 µg/mL); especially, a3 presented significantly prominent inhibitory activity with an EC50 of 0.027 µg/mL. In the pursuit of further expanding the antifungal spectrum of cryptolepine, ring-opened compound f1 produced better activity with an EC50 of 3.632 µg/mL against R. solani and an EC50 of 5.599 µg/mL against F. graminearum. Furthermore, a3 was selected to be a candidate to investigate its preliminary antifungal mechanism to B. cinerea, revealing that not only spore germination was effectively inhibited and the normal physiological structure of mycelium was severely undermined but also detrimental reactive oxygen was obviously accumulated and the normal function of the nucleus was fairly disordered. Besides, in vivo curative experiment against B. cinerea found that the therapeutic action of a3 was comparable to that of the positive control azoxystrobin. These results suggested that compound a3 could be regarded as a novel and promising agent against B. cinerea for its valuable potency.

Bioassay-guided isolation of two antifungal compounds from Magnolia officinalis, and the mechanism of action of honokiol.

Magnolia officinalis, as a well-known herb worldwide, has been widely used to treat multiple diseases for a long time. In this study, the petroleum ether extract from M. officinalis showed effective antifungal activity against seven plant pathogens (particularly against R. solani with an inhibition rate of 100.00% at 250 µg/mL). Honokiol and magnolol, isolated by the bioassay-guided method, exhibited greater antifungal activity than tebuconazole (EC50 = 3.07 µg/mL, p ≤ 0.001) against R. solani, which EC50 values were 2.18 µg/mL and 3.48 µg/mL, respectively. We used transcriptomics to explore the mechanism of action of honokiol against R. solani. Results indicated that honokiol may exert antifungal effects by blocking the oxidative phosphorylation metabolic pathway. Further studies indicated that honokiol induced ROS overproduction, disrupted the mitochondrial function, affected respiration, and blocked the TCA cycle, which eventually inhibited ATP production. Besides, honokiol also damaged cell membranes and caused morphological changes. This study demonstrated that the lignans isolated from M. officinalis possess the potential to be developed as botanical fungicides.

[Effects of Huoxiang Zhengqi liquid on enteric mucosal immune responses in mice with Bacillus dysenteriae and Salmonella typhimurium induced diarrhea].

OBJECTIVE: To explore the effects of Huoxiang Zhengqi liquid (HXZQ) on enteric mucosal immune responses in mice with Bacillus dysenteriae and Salmonella typhimurium induced diarrhea (BSD). METHOD: Mice were randomly divided into four groups with 10 mice in each group: control group (control), BSD group, Huoxiang Zhengqi liquid treated BSD groups at high dosage and low dosage (HXZQ high, HXZQ low). HXZQ was administrated from the day of diarrhea induction at dosage of 5.21 g kg(-1) and 0.52 g kg (-1) respectively. Peyer's patch and periphery lymphocytes were prepared for flow cytometry, and level of TNF-alpha in periphery and enteric tissue homogenate were determined with ELISA. Student's t-test was used for statistics. RESULT: Mice in BSD group started showing continuous diarrhea at the day of induction till the fourth day when the mice were sacrificed. Diarrhea in the mice of HXZQ high and low groups lasted for 36 and 54 h respectively. There were more CD4+ and CD8+ cells in periphery, less CD4+ cells in peyer's patch in BSD mice comparing to normal mice. In peyer's patch, there were more CD8+ cells in mice in HXZQ high and low groups and more CD4+ in mice in HXZQ high group. Higher level TNF-alpha in periphery and intestinal tissue homogenate in BSD group were observed. Mice in HXZQ high group showed the decreased level TNF-alpha in periphery and enteric tissue homogenate. CONCLUSION: The immune regulation on peyer's patch CD4+ and CD8+ cells and suppression on TNF-alpha level in enteric homogenate might partially explain the effect of HXZQ on improvement of BSD.

Effect of Gui Zhi decoction on enteric mucosal immune in mice with collagen-induced arthritis.

AIM: To explore the effect of Gui Zhi decoction on enteric mucosal immune in type II collagen-induced arthritis (CIA) in DBA mice. METHODS: Eighty DBA/1, weighing 18-22 g, were randomly divided into four groups with 20 in each group: control group, CIA group, treatment groups at high dosage and low dosage (GZH and GZL). CIA was induced by immunization with type II collagen (CII) emulsified with equal complete adjuvant at 0.1 mg CII each mouse. Blood lymphocyte suspension was screened for CD4 and CD8 expression using a flow cytometry, the CD4 and CD8 and secretory IgA (sIgA)-positive cells in enteric lamina propria tested with immunohistochemical staining. Tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1)-beta, and IL-6 concentrations in serum were assayed with RIA. RESULTS: Gui Zhi decoction can lower the arthritic scores and decrease the occurrence of arthritis. The CD4, CD8, and sIgA-positive cells in CIA mice are less than in control mice, and in Gui Zhi decoction at high dosage could restore the lowered CD4- and CD8-positive cells in lamina propria, and at both high and low dosages could increase the lowered sIgA-positive cells in lamina propria, even still lower than in normal mice. In periphery, the CD4 cells in periphery are higher in CIA mice than in control mice, and Gui Zhi decoction at high and low dosages could decrease the CD4 and CD8 cells. Also, Gui Zhi decoction at high dosage could decrease the IL-6 and TNF-alpha concentration in serum. CONCLUSION: Gui Zhi decoction can lower the arthritic scores and decrease the incidence of CIA in mice, and the mechanism is in part regulating enteric mucosal immune.

[Antioxidant and anti-inflammatory effects of cyanidin from cherries on rat adjuvant-induced arthritis].

OBJECTIVE: To assess the possible antioxidant and anti-inflammatory activity of cyanidin from cherries on adjuvant induced arthritis (AA) in SD rats. METHOD: Arthritis was induced by the complete Freud's adjuvant in male Sprague Dauley rats and assessed based on paw swelling. Rats were randomly divided into normal group (NM), adjuvant arthritis group (AA) and three cyanidin-treated groups in high dosage (HA), middle dosage (MA), and low dosage (LA). The morphological changes in the hind limbs were conducted under a light microscope. We detected glutathione (GSH) in whole blood and malonaldehyde (MDA), superoxide dismutase (SOD), total antioxidative capacity (T-AOC) activity in serum by special kits to assess the antioxidant effects of cyanidin on AA. Moreover, the prostaglandin E2 (PGE2) levels in paw tissues were determined by radioimmunoassay and TNF-alpha levels in serum were determined using ELISA kits specific for rat. RESULT: The cyanidin could protect against the paws swelling in AA rats. From the day 14 after AA induction, the swellings of the cyanidin treated groups at high dosage and low dosage were significantly reduced compared with the model group (P < 0.05, 0.01). Histological examination of sections through the hind limbs revealed alleviation of inflammatory reaction in the joint after the treatment. The cyanidin at high and low dosage could increase the GSH, SOD activity and T-AOC levels in whole blood or serums and decrease MDA in AA rats (P < 0.01). The cyanidin could decrease the PGE2 levels in paw tissues and the TNF-alpha levels in serum at high and low dosages (P < 0.01). CONCLUSION: The cyanidin could protect against the paws swelling in AA rats, and alleviate the inflammatory reaction in the joint, and the mechanism might be via the increase activity of GSH, SOD and T-AOC that improve the total antioxidative capacity and scavenge the free radicals, perhaps as a result of that the levels of the PGE2 in paw tissues and TNF-alpha contents in serum were decreased. The results suggest that the cyanidin from cherries could be one of the potential candidates for the alleviation of arthritis.

Protection of human lung cells against hyperoxia using the DNA base excision repair genes hOgg1 and Fpg.

Hyperoxia causes pulmonary toxicity in part by injuring alveolar epithelial cells. Previous studies have shown that toxic oxygen-derived species damage DNA and this damage is recognized and repaired by either human enzyme 8-oxoguanine DNA glycosylase (hOgg1) or Escherichia coli enzyme formamidopyrimidine DNA glycosylase (Fpg). To determine whether these DNA repair proteins can reduce O(2)-mediated DNA damage in lung cells, A549 lung epithelial cells were transduced with either hOgg1 or Fpg using a retroviral vector containing enhanced green fluorescent protein. Expression of each gene in the transduced cells was confirmed by fluorescent microscopy, Northern blotting, Western blotting, and an enzymatic oligonucleotide cleavage assay. A549 cells expressing either hOgg1 or Fpg were protected from hyperoxia as evidenced by a decrease in DNA damage and a corresponding increase in cell survival. Further, we determined that overexpression of hOgg1 or Fpg partially mitigated the toxic effects of hydrogen peroxide in lung cells. Our data suggest that increased expression of DNA base excision repair genes might represent a new approach for protecting critical lung cells from the toxic effects of hyperoxia.

Escherichia coli FPG and human OGG1 reduce DNA damage and cytotoxicity by BCNU in human lung cells.

The pulmonary complications of 1,3-N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU) are among the most important dose-limiting factors of BCNU-containing cancer chemotherapeutic regimens. BCNU damages DNA of both cancer cells and normal cells. To increase the resistance of lung cells to BCNU, we employed gene transfer of Escherichia coli formamidopyrimidine-DNA glycosylase (FPG) and human 8-oxoguanine-DNA glycosylase (hOGG1) to A549 cells, a lung epithelial cell line, using a bicistronic retroviral vector, pSF91-RE, that encoded both FPG/hOGG1 and an enhanced green fluorescent protein. The transduced epithelial cells were sorted by flow cytometry, and expression of FPG/hOGG1 protein was determined by the level of FPG/hOGG1 RNA and enzyme activity. The single-cell gel electrophoresis (comet assay) measured DNA damage induced by BCNU. FPG/hOGG1-expressing A549 cells incubated with 40-500 microg/ml BCNU exhibited significantly less DNA damage than vector-transduced cells. In addition, FPG- and/or hOGG1-expressing cells incubated with 10-40 microg/ml BCNU showed at least a 25% increase in cell survival. Gene transfer of FPG/hOGG1 reduced BCNU-induced DNA damage and cytotoxicity of cultured lung cells and may suggest a new mechanism to reduce BCNU pulmonary toxicity.