Enhancing the biosynthesis of nicotinamide mononucleotide in Lactococcus lactis by heterologous expression of FtnadE.

BACKGROUND: Nicotinamide mononucleotide (NMN), a key intermediate of nicotinamide adenine dinucleotide, plays an important in anti-aging and disease. Lactococcus lactis, an important probiotic lactic acid bacteria (LAB), has shown great potential for the biosynthesis of NMN, which will significantly affect the probiotic effects of the dairy products. RESULTS: We used the CRISPR/nCas9 technique to knockout nadR gene of L. lactis NZ9000 to enhance the accumulation of NMN by 61%. The nadE* gene from Francisella tularensis with codon optimization was heterologous in L. lactis NZ9000ΔnadR and has a positive effect on NMN production. Combined with optimization of the concentration of substrate nicotinamide, a final intracellular NMN titer was 2289 µmol L-1  mg-1 with 10 g L-1 nicotinamide supplement, which was 5.7-fold higher than that of the control. The transcription levels of key genes (pncA, nadD and prs1) involved in NMN biosynthesis were up-regulated by more than two-fold, indicating that the increase of NMN titer was attributed to FtnadE* heterologous expression. CONCLUSION: Our study provides a better understanding of the NMN biosynthesis pathway in L. lactis, and can facilitate NMN production in LAB via synthetic biology approaches. © 2022 Society of Chemical Industry.

Berberine attenuates sodium palmitate-induced lipid accumulation, oxidative stress and apoptosis in grass carp(Ctenopharyngodon idella)hepatocyte in vitro.

The objective of this work was to investigate the effect of berberine (BBR) on the Cell viability, lipid accumulation, apoptosis, cytochrome c, caspase-9 and caspase-3 in lipid accumulation-hepatocytes induced by sodium palmitate in vitro. The lipid accumulation-hepatocytes (induced by 0.5 mM sodium palmitate for 24 h) were treated with 5 µM berberine for 12 h. Then, the Cell viability, intracellular triglyceride (TG) content, lipid peroxide (LPO), malonaldehyde (MDA) content, cytochrome c, caspase-9, caspase-3 and apoptosis were detected. Sodium palmitate decreased Cell viability and increased intracellular TG content, lipid droplet accumulation, LPO and MDA concentrations, caused caspase-3 and caspase-9 activation, then led to apoptosis accompanied by cytochrome c release from mitochondria into the cytoplasm. Beberine could improve intracellular lipid droplet accumulation and oxidative stress, while reduce apoptosis induced by sodium palmitate.

Growth performance, innate immune responses and disease resistance of fingerling blunt snout bream, Megalobrama amblycephala adapted to different berberine-dietary feeding modes.

A 8-week feeding trial was conducted to evaluate the effect of different berberine-dietary feeding modes on growth, non-specific immune responses and disease resistance of blunt snout bream, Megalobrama amblycephala. Fish (average initial weight 4.70 ± 0.02 g) were fed two fat levels (5% and 10%) diets in three berberine-feeding modes (supplementing 50 mg/kg berberine continuously, two-week or four-week intervals) with four replicates, respectively. Then, fish were challenged by Aeromonas hydrophila and mortality was recorded for the next 96 h after feeding trial. The results showed that different feeding modes of berberine significantly influenced growth, innate immunity and antioxidant capability of fish. Fish fed normal diet with 50 mg/kg berberine at two-week interval mode reflected remarkably (P < 0.05) high weight gain (WG). Plasma TC and TG contents were significantly (P < 0.05) decreased. The lysozyme (LYZ) activities, complement component 3 (C3) and complement component 4 (C4) concentrations were significantly (P < 0.05) increased. Fish not only exhibited relatively low hepatopancreas malondialdehyde (MDA) and lipid peroxide (LPO) contents, but also significantly (P < 0.05) improved superoxide dismutase (SOD) and catalase (CAT) activities. Fish mortality after challenged by Aeromonas hydrophila was decreased. Same results were also presented in fish fed high-fat diet with 50 mg/kg berberine at two-week, four-week intervals or continuous feeding modes. Based on fish healthy improvement and feeding cost saving, blunt snout bream fed normal diet with 50 mg/kg berberine at two-week interval or fed high-fat diet with berberine at two-week or four-week intervals were optimal feeding mode, respectively.

Berberine attenuates oxidative stress and hepatocytes apoptosis via protecting mitochondria in blunt snout bream Megalobrama amblycephala fed high-fat diets.

High-fat diets may have favorable effects on growth and cost, but high-fat diets often induce excessive fat deposition, resulting in liver damage. This study aimed to identify the hepatoprotective of a Chinese herb (berberine) for blunt snout bream (Megalobrama amblycephala). Fish were fed with a normal diet (LFD, 5 % fat), high-fat diet (HFD, 15 % fat) or berberine-supplemented diets (BSD, 15 % fat with berberine 50 or 100 mg/kg level) for 8 weeks. After the feeding, histology, oxidative status and mitochondrial function of liver were assessed. The results showed that HFD caused fat accumulation, oxidative stress and apoptosis in hepatocytes of fish. Hepatocytes in HFD group appeared to be hypertrophied, with larger liver cells diameter than these of LFD group. Berberine-supplemented diets could attenuate oxidative stress and hepatocytes apoptosis. HFD induced the decreasing mitochondrial complexes activities and bulk density and surface area density. Berberine improved function of mitochondrial respiratory chain via increasing the complex activities. Moreover, the histological results showed that berberine has the potential to repair mitochondrial ultrastructural damage and elevate the density in cells. In conclusion, our study demonstrated that berberine has attenuated liver damage induced by the high fat mainly via the protection for mitochondria.

Effects of berberine on the growth and immune performance in response to ammonia stress and high-fat dietary in blunt snout bream Megalobrama amblycephala.

This study aimed to figure out the effects of berberine on growth performance, immunity, oxidative stress and hepatocyte apoptosis of blunt snout bream (Megalobrama amblycephala) fed with high-fat diet. 320 fish (80.00 ± 0.90 g) were divided randomly into four trial groups (each with four replicates) and fed with 4 diets (normal diet, normal diet with 50 mg/kg berberine, high-fat diet, high-fat diet with 50 mg/kg berberine), respectively. At the end of the feeding trial, ammonia stress test was carried out for 5 days. The result showed the growth performance, immune parameters including plasm acid phosphatase (ACP) activities, lysozyme (LYZ) activities and alternative complement C3 and C4 contents were suppressed in fish fed with high-fat diets but improved in berberine diets compared with control (normal diet). Hepatopancreas oxidative status, the malondialdehyde (MDA), protein carbonyl (PC) and lipid peroxide (LPO) were increased significantly (P < 0.05) when fish were fed with high-fat diets. Berberine could slow the progression of the oxidative stress induced by high-fat through increasing superoxide dismutase (SOD) activities and total sulfydryl (T-SH) levels of fish. And the hepatocyte apoptosis in the high-fat group could also be alleviated by berberine. After the ammonia stress test, the accumulative mortality was extremely (P < 0.05) low in fish fed high-fat diet with berberine compared to other groups. It was concluded berberine as a functional feed additive significantly inhibited the progression of oxidative stress, reduced the apoptosis and enhanced the immunity of fish fed with high-fat diet.

Identification and differential expression of hepatopancreas microRNAs in red swamp crayfish fed with emodin diet.

Using high-throughput Illumina Solexa system, the differential miRNA expressions from hepatopancreas in red swamp crayfish (Procambarus clarkii) fed with diets containing 0 (control) and 75 mg emodin kg(-1) (trial) were identified, respectively. As a result, 13,335,928 raw reads from the control sample and 14,938,951 raw reads from the trial sample were obtained while 13,053,344 (98.77%) and 14,517,522 (98.34%) small RNA were identified, respectively. 106 mature miRNAs (belonging to 68 miRNA gene families) were identified. 35 miRNAs displayed significantly differential expressions between two libraries. Of these, comparing to the control library, 6 miRNAs were significantly up-regulated and 29 miRNAs were significantly down-regulated. Moreover, 5 novel miRNAs (2 from control sample, 3 from trial sample) and target genes were predicted. GO analysis suggested that these miRNAs might be involved in innate immune response, growth, metabolism, cellular process, biological regulation and stimulus response. Our knowledge from this study could contribute to a better understanding of the miRNAs roles in regulating innate immune response and the study of miRNA function in crayfish.

Absence of fks1p in lager brewing yeast results in aberrant cell wall composition and improved beer flavor stability.

The flavor stability during storage is very important to the freshness and shelf life of beer. However, beer fermented with a yeast strain which is prone to autolyze will significantly affect the flavor of product. In this study, the gene encoding ß-1,3-glucan synthetase catalytic subunit (fks1) of the lager yeast was destroyed via self-clone strategy. ß-1,3-glucan is the principle cell wall component, so fks1 disruption caused a decrease in ß-1,3-glucan level and increase in chitin level in cell wall, resulting in the increased cell wall thickness. Comparing with wild-type strain, the mutant strain had 39.9 and 63.41 % less leakage of octanoic acid and decanoic acid which would significantly affect the flavor of beer during storage. Moreover, the results of European Brewery Convention tube fermentation test showed that the genetic manipulation to the industrial brewing yeast helped with the anti-staling ability, rather than affecting the fermentation ability. The thiobarbituric acid value reduced by 65.59 %, and the resistant staling value increased by 26.56 %. Moreover, the anti-staling index of the beer fermented with mutant strain increased by 2.64-fold than that from wild-type strain respectively. China has the most production and consumption of beer around the world, so the quality of beer has a significant impact on Chinese beer industry. The result of this study could help with the improvement of the quality of beer in China as well as around the world.

Combined effects of dietary fructooligosaccharide and Bacillus licheniformis on innate immunity, antioxidant capability and disease resistance of triangular bream (Megalobrama terminalis).

This study was conducted to investigate the effects of fructooligosaccharide (FOS) and Bacillus licheniformis (B. licheniformis) and their interaction on innate immunity, antioxidant capability and disease resistance of triangular bream Megalobrama terminalis (average initial weight 30.5 ± 0.5 g). Nine experimental diets were formulated to contain three FOS levels (0, 0.3% and 0.6%) and three B. licheniformis levels (0, 1 × 10(7), 5 × 10(7) CFU g(-1)) according to a 3 × 3 factorial design. At the end of the 8-week feeding trial, fish were challenged by Aeromonas hydrophila (A. hydrophila) and survival rate was recorded for the next 7 days. The results showed that leucocyte counts, alternative complement activity as well as total serum protein and globulin contents all increased significantly (P < 0.05) as dietary B. licheniformis levels increased from 0 to 1 × 10(7) CFU g(-1), while little difference (P > 0.05) was observed in these parameters in terms of dietary FOS levels. Both plasma alkaline phosphatase and phenoloxidase activities were significantly (P < 0.05) affected only by dietary FOS levels with the highest values observed in fish fed 0.6 and 0.3% FOS, respectively. Both immunoglobulin M content and liver superoxide dismutase (SOD) activity were significantly affected (P > 0.05) by both FOS and B. licheniformis. Liver catalase, glutathione peroxidase as well as plasma SOD activities of fish fed 1 × 10(7) CFU g(-1)B. licheniformis were all significantly (P < 0.05) higher than that of the other groups, whereas the opposite was true for malondialdehyde content. After A. hydrophila challenge, survival rate was not affected (P > 0.05) by either FOS levels or B. licheniformis contents, whereas a significant (P < 0.05) interaction between these two substances was observed with the highest value observed in fish fed 0.3% FOS and 1 × 10(7) CFU g(-1)B. licheniformis. The results of this study indicated that dietary FOS and B. licheniformis could significantly enhance the innate immunity and antioxidant capability of triangular bream, as well as improve its disease resistance. The best combination of these two prebiotics and/or probiotics was 0.3% FOS and 1 × 10(7) CFU g(-1)B. licheniformis.

Dietary carbohydrate/lipid ratios affect stress, oxidative status and non-specific immune responses of fingerling blunt snout bream, Megalobrama amblycephala.

This study aimed to evaluate the effects of dietary carbohydrate/lipid (CHO:L) ratios on stress, liver oxidative status and non-specific immune responses of fingerling blunt snout bream. Fish were fed six isonitrogenous and isoenergetic diets containing various CHO:L ratios for 10 weeks. After the feeding trial, fish were challenged by Aeromonas hydrophila and survival rate was recorded for the next 10 days. The lowest plasma cortisol, lactate, aspartate aminotransferase and alanine aminotransferase were all observed in fish fed a CHO:L ratio of 5.64. They were significantly (P < 0.05) lower than those of fish offered the lowest CHO:L ratio, but showed little difference (P > 0.05) with those of fish fed the highest CHO:L ratio. This also held true for liver catalase and glutathione peroxidase activities, whereas the opposite was true for liver reduced glutathione contents, plasma lysozyme and alternative complement (ACH50) activities. Contrary to leucocyte counts, plasma glucose levels, liver malondialdehyde contents, blood haemoglobin contents and erythrocyte numbers all increased significantly (P < 0.05) with decreasing dietary CHO:L ratios. The highest plasma total protein and globulin content both observed in fish fed a CHO:L ratio of 2.45 was significantly (P < 0.01) higher than that of fish offered the lowest CHO:L ratio, but showed no statistical difference (P > 0.05) with that of the other groups. After challenge, fish fed the lowest CHO:L ratio obtained significantly (P < 0.05) low survival rate. However, survival rate showed little difference (P > 0.05) as dietary CHO:L ratios ranged from 3.67 to 24.20. The results of this study indicated that high dietary lipid may cause metabolic stress of fingerling blunt snout bream, as might consequently lead to the elevated liver oxidation rates, impaired liver function, depressed immunity and reduced resistance to A. hydrophila infection of this species, whereas the opposite was true for carbohydrate enriched diets.

[Study on volatile components from peel of Aquilaria sinensis and the anti-tumor activity].

OBJECTIVE: To study the volatile constituents from peel of Aquilaria sinensis (Lour.) Gilg. and its anti-tumor activity. METHODS: The volatile components from peel of A. sinensis were extracted by chloroform and analysed by the GC-MS technique. Assay the peel extract of A. sinensis on MCF-7 cell proliferation using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) method. RESULTS: GC-MS gave 131 peaks and 28 compounds have been identified. The contents of the benzene compounds accounted 7.79% of the volatile components, the esquiterpenes accounted 5.44%, and the relative content of fatty acids accounted 3.08%. In addition, two 2-(2-phenylethyl)-color ketones and a chromone component were detected, and the total relative content accounted 12.3%. The inhibition rate of human breast cancer cells reached 99. 6% when the concentration was 500 microg/mL. CONCLUSION: The volatile components from peel of A. sinensis were analysed and a large number of characteristic elements of A. sinensis were detected for the first time. The chloroform extract from peel of A. sinensis significantly inhibited the proliferation of human breast cancer cells.

1,3-alternate and partial cone conformers of tetramethyl (5,11,17,23-tetra-tert-butyl-2,8,14,20-tetrathiacalix[4]arene-25,26,27,28-tetrayltetraoxy)tetraacetate and tetramethyl (5,11,17,23-tetrabromo-2,8,14,20-tetrathiacalix[4]arene-25,26,27,28-tetrayltetraoxy)tetraacetate.

The crystal structures of the two title thiacalix[4]arene derivatives, C52H64O12S4, (I), and C36H28Br4O12S4, (II), are reported. Compound (I) has crystallographic C2 symmetry and adopts a 1,3-alternate conformation where the four -OCH2CO2Me groups are located alternately above and below the virtual plane (R) defined by the four bridging S atoms. The dihedral angles between the plane (R) and the aromatic rings are 87.17 (7) and 87.60 (8) degrees . Compound (II) has a partial cone conformation in which the pendant -OCH2CO2Me group of the rotated aryl ring is oriented away from the thiacalixarene cavity formed by the other three aryl rings. The dihedral angles between the plane (R) and the aryl rings range from 17.47 (10) to 85.98 (6) degrees . In the supramolecular structure of (II), the molecular components are linked into a two-dimensional framework by a combination of C-H...O hydrogen bonds and C-Br...C interactions. This study demonstrates the usefulness of these motif-generating interactions and thiacalix[4]arene derivatives in crystal engineering.

Molecular characterization of microtubule-associated protein 1-light chain 3B in Megalobrama amblycephala fed with high fat/berberine diets.

This study tested the effect of berberine on autophagy-related protein of Megalobrama amblycephala fed with high fat diet under different feeding modes. The full-length complementary DNA (cDNA) of microtubule-associated protein 1-light chain 3B (LC3B) was 1871 bp with an open reading frame of 378 bp encoding 125 amino acids. High homology at nucleotide and amino acid sequences to carp LC3B was revealed though sequence analysis. LC3B was mainly (P < 0.05) expressed in hepatopancreas but lower in several peripheral tissues, including gill, intestine, kidney, and spleen. The fish (average initial weight 4.70 ± 0.02 g) were fed with eight experiment diets containing two lipid levels (5 and 10%) or four berberine-feeding modes (without berberine, supplementing 50 mg/kg berberine at 2-week intervals, 4-week intervals, or continuous) for 8 weeks. The results showed that the numbers of autophagosomes and hepatopancreas LC3B messenger RNA (mRNA) expression levels were significantly (P < 0.05) affected both by dietary lipid level and beberine feeding mode, and the highest (P < 0.05) numbers of autophagosomes and LC3B expression levels were observed in fish at berberine continuous feeding mode groups. The findings may provide the molecular mechanisms underlying lipid metabolism and immune effect of berberine, which was associated with enhanced autophagy in fish.

1-(2-Methoxy-ethoxy)-4-nitro-benzene.

The title compound, C(9)H(11)NO(4), is an inter-mediate for dyes and drugs. The O-C-C-O chain adopts a synclinal conformation. The crystal structure is stabilized by C-H⋯O hydrogen bonds.

Molecular characterization of carnitine palmitoyltransferase IA in Megalobrama amblycephala and effects on its expression of feeding status and dietary lipid and berberine.

Carnitine palmitoyltransferase I (CPT I, EC 2.3.1.21) controls the main regulatory step of fatty acid oxidation, and hence studies of its molecular characterization are useful to understand lipid metabolism in cultured fish. Here, a full-length cDNA coding CPT I was cloned from liver of blunt snout bream Megalobrama amblycephala. This cDNA obtained covered 2499bp with an open reading frame of 2181bp encoding 726 amino acids. This CPT I mRNA predominantly expressed in heart and white muscle, while little in eye and spleen. The phylogenetic tree constructed on the basis of sequence alignments among several vertebrate species suggests that this blunt snout bream CPT I sequence belongs to the CPT IA family. In order to investigate the characterization of CPT IA mRNA expression, post-prandial experiment and feeding trial were conducted. The results showed that CPT IA mRNA expression was unchanged from 2 to 12h, and then significantly increased at 24h post-feeding in liver and heart. Berberine, an alkaloid, was identified as a promising lipid-lowering drug. In order to elucidate the effect of berberine on CPT I expression, fish were fed for 8 weeks with three diets (low-fat diet (LFD, 5% fat), high-fat diet (HFD, 15% fat), and berberine-supplemented diet (BSD, 15% fat). The results showed that HFD could decrease the expression of CPT IA and PPARα, while BSD increased those expressions.

Cloning and characterization of microsomal triglyceride transfer protein gene and its potential connection with peroxisome proliferator-activated receptor (PPAR) in blunt snout bream (Megalobrama amblycephala).

Microsomal triglyceride transfer protein (MTTP), a major intracellular protein capable of transferring neutral lipids, plays a pivotal role in the assembly and secretion of apolipoprotein B-containing lipoproteins. In this study, MTTP cDNA was firstly cloned from the liver of blunt snout bream (Megalobrama amblycephala), the full-length cDNA covered 3457-bp with an open reading frame of 2661-bp, which encodes 886 amino acids, including a putative signal peptide of 24 amino acids long. After the feeding trial, a graded tissue-specific expression pattern of MTTP was observed and high expression abundance in the liver and intestine indicated its major function in lipid transport in this fish species. In addition, expression of genes encoding MTTP as well as peroxisome proliferator-activated receptor (PPAR), which are transcription factors and serve as key regulators in lipid homoeostasis, was all affected by dietary lipid and choline supplementations. Elevated dietary lipid levels significantly increased the liver, intestinal and muscle MTTP mRNA abundance. Additionally, the down-regulation of MTTP expression in the liver and muscle was observed when fish were fed with inadequate choline supplementation in high-fat diet, yet up-regulated as supplementing extra choline in diet. Expressions of PPARα and PPARß in the liver and muscle showed similar trend of MTTP expression. The results suggested the potential connection of MTTP and PPAR in response to different dietary nutritional factors. Furthermore, extra choline supplementations could promote lipid transfer and enhance fatty acid oxidation, which indicated a molecular mechanism of choline on diminishing fat accumulation in blunt snout bream.

Association of mitochondrial dysfunction with oxidative stress and immune suppression in Blunt Snout Bream Megalobrama amblycephala fed a high-fat diet.

High-fat diets may have favorable effects on growth, partly based on protein sparing, but high-fat diets often lead to fatty liver (excessive fat deposition in the liver), which may be deleterious to fish growth and health. The goal of this study was therefore to investigate possible adverse effects and how they develop. Juvenile Blunt Snout Bream Megalobrama amblycephala (initial weight ± SE = 17.70 ± 0.10 g) were fed two diets (5% fat [control] or 15% fat). After 8 weeks, fish that were fed the 15% fat diet showed a high rate of mortality and poor growth. The histological results clearly showed that the high fat intake resulted in fat and glycogen accumulation and structural alterations of the hepatocytes, mitochondria, and nuclei. In the high-fat group, impairments of the mitochondria included mitochondrial swelling and the loss of cristae and matrix. Fish that were given the 15% fat diet exhibited low succinate dehydrogenase and Na(+),K(+)-ATPase activities and increased cytochrome-c release from the mitochondria. Expression of genes for complex I and III subunits of the mitochondrial respiratory chain were down-regulated in fish that received the high-fat diet. Increases in malondialdehyde level and the ratio of oxidized glutathione to reduced glutathione suggested oxidative stress in the livers of fish from the high-fat diet group. Moreover, the lower leukocyte count, lysozyme and alternative complement activities, and globulin level in fish that received the high-fat diet indicated suppressive immune responses. Overall, the intake of excessive fat impaired mitochondrial bioenergetics and physiological functions. The dysfunction of the mitochondria subsequently mediated oxidative stress and hepatocyte apoptosis, which in turn led to the reduced efficacy of the immune system.

Hepatic ß-oxidation and regulation of carnitine palmitoyltransferase (CPT) I in blunt snout bream Megalobrama amblycephala fed a high fat diet.

High-fat diets may promote growth, partly through their protein-sparing effects. However, high-fat diets often lead to excessive fat deposition, which may have a negative impact on fish such as poor growth and suppressive immune. Therefore, this study investigated the effects of a fat-rich diet on the mechanisms of fat deposition in the liver. Three-hundred blunt snout bream (Megalobrama amblycephala) juveniles (initial mass 18.00 ± 0.05 g) were fed with one of two diets (5% or 15% fat) for 8 weeks. ß-Oxidation capacity and regulation of rate-limiting enzymes were assessed. Large fat droplets were present in hepatocytes of fish fed the high-fat diet. This observation is thought to be largely owing to the reduced capacity for mitochondrial and peroxisomal ß-oxidation in the livers of fish fed the high-fat diet, as well as the decreased activities of carnitine palmitoyltransferase (CPT) I and acyl-CoA oxidase (ACO), which are enzymes involved in fatty-acid metabolism. Study of CPT I kinetics showed that CPT I had a low affinity for its substrates and a low catalytic efficiency in fish fed the high-fat diet. Expression of both CPT I and ACO was significantly down-regulated in fish fed the high-fat diet. Moreover, the fatty-acid composition of the mitochondrial membrane varied between the two groups. In conclusion, the attenuated ß-oxidation capacity observed in fish fed a high-fat diet is proposed to be owing to decreased activity and/or catalytic efficiency of the rate-limiting enzymes CPT I and ACO, via both genetic and non-genetic mechanisms.

Trichlorfon-induced apoptosis in hepatocyte primary cultures of Carassius auratus gibelio.

Trichlorfon, an organophosphorus pesticide, can disrupt metabolism, reproduction and immune functions of some aquatic animals. In the present study, the effect of trichlorfon on apoptosis and the underlying apoptotic mechanism were investigated in primary cultures of Carassius auratus gibelio hepatocytes. Analyses of cultures exposed to 0, 0.01, 0.1, and 1.0 mg L(-1) trichlorfon concentrations for 24h indicated that trichlorfon induced apoptosis and caused nuclear shrinkage, cell membrane rupture, cytoskeletal collapse, loss of cytoplasm, mitochondria vacuolization, and apoptotic body formation, as well as lipid droplet accumulation. Trichlorfon increased intracellular reactive oxygen species and malondialdehyde concentrations and caused cytochrome c release from mitochondria into the cytoplasm, leading to caspase-3 activation. These findings contributed to a better understanding of the mechanisms underlying trichlorfon-induced apoptosis via activation of mitochondrial pathways while clearly indicating that trichlorfon-induced cell death was via apoptosis accompanied by mitochondrial cytochrome c release and consequent caspase-3 activation.