An overview of aflatoxin B1 biotransformation and aflatoxin M1 secretion in lactating dairy cows

Milk is considered a perfect natural food for humans and animals. However, aflatoxin B1 (AFB1) contaminating the feeds fed to lactating dairy cows can introduce aflatoxin M1 (AFM1), the main toxic metabolite of aflatoxins into the milk, consequently posing a risk to human health. As a result of AFM1 monitoring in raw milk worldwide, it is evident that high AFM1 concentrations exist in raw milk in many countries. Thus, the incidence of AFM1 in milk from dairy cows should not be underestimated. To further optimize the intervention strategies, it is necessary to better understand the metabolism of AFB1 and its biotransformation into AFM1 and the specific secretion pathways in lactating dairy cows. The metabolism of AFB1 and its biotransformation into AFM1 in lactating dairy cows are drawn in this review. Furthermore, recent data provide evidence that in the mammary tissue of lactating dairy cows, aflatoxins significantly increase the activity of a protein, ATP-binding cassette super-family G member 2 (ABCG2), an efflux transporter known to facilitate the excretion of various xenobiotics and veterinary drugs into milk. Further research should focus on identifying and understanding the factors that affect the expression of ABCG2 in the mammary gland of cows.     

Expression and subcellular localization of efflux transporter ABCG2/BCRP in important tissue barriers of lactating dairy cows,sheep and goats

Expression of efflux transporter ABCG2/BCRP in tissues barriers has shown to be associated with altered pharmaco‐ and toxicokinetics of xenobiotics. Until now, little is known about the functional expression of this transporter in dairy animals. We therefore systematically examined the expression and subcellular localization of ABCG2/BCRP in small intestine, colon, lung, liver, kidney and mammary gland in lactating cows, sheep and goats. Carrier expression was investigated by RT‐PCR and Western blot analysis showing highest expression of ABCG2/BCRP in small intestine and mammary gland, high levels in liver and moderate amounts of protein in lung, colon and kidney. Regarding subcellular localization, BCRP was predominantly found at the apical plasma membrane of small intestine, colon, bronchial epithelium, bile ducts and overall in endothelial structures in all tested species. In the mammary gland, there was strong apical staining of the alveolar epithelial cells and most of the ducts in all dairy ruminants. We also detected significantly elevated protein expression in lactating mammary gland compared with nonlactating cows, sheep and goats. Our results contribute to the role of BCRP in cytoprotection and disposition in important tissue barriers and may have important implications for veterinary pharmacotherapy of dairy animals using drugs identified as BCRP substrates.     

Interaction of enrofloxacin with breast cancer resistance protein (BCRP/ABCG2): influence of flavonoids and role in milk secretion in sheep

The ATP-binding cassette (ABC) transporter breast cancer resistance protein (BCRP)/ABCG2 is a high-capacity efflux transporter with wide substrate specificity located in apical membranes of epithelia, which is involved in drug availability. BCRP is responsible for the active secretion of clinically and toxicologically important substrates to milk. The present study shows BCRP expression in sheep and cow by immunoblotting with MAb (BXP-53). Vanadate-sensitive ATPase activity with specific BCRP substrates and inhibitors was measured in bovine mammary gland homogenates. To assess the role of BCRP in ruminant mammary gland we tested the fluoroquinolone enrofloxacin (ENRO). In polarized cell lines, ENRO was transported by Bcrp1/BCRP with secretory/absorptive ratios of 6.5 and 2 respectively. The efflux was blocked by the BCRP inhibitor Ko143. ENRO pharmacokinetics in plasma and milk was studied in sheep after co-administration of drug (2.5 mg/kg, i.v.) and genistein (0.8 mg/kg, i.m.) or albendazole sulfoxide (2 mg/kg, i.v) as BCRP inhibitors. Concomitant administration of BCRP inhibitors with ENRO had no significant effect on the plasma disposition kinetics of ENRO but decreased ENRO concentrations in milk.     

Investigation of the metabolic activity of a bovine mammary epithelial cell line (BME-UV1)

The metabolic activity of a mammary epithelial cell line (BME-UV1) was evaluated on monolayers exposed, in serum free medium, to different concentrations (2-4-8 muM) of aflatoxin B1 (AFB1), a mycotoxin eliminated into milk especially as hydroxylated metabolite aflatoxin M1 (AFM1). After 4, 8, 12, 24 h of treatment, a dose and time dependent production of AFM1 has been detected. As the enzymes involved in the hydroxylation of AFB1 in bovine hepatocytes are mainly CYP1A and CYP3A, the results suggest that BME-UV1 express CYP450 isoenzymes which metabolize AFB1 thus representing a potential model for the investigation of the metabolic activity of bovine mammary epithelial tissue.     

Analysis of the effect of the bovine adenosine triphosphate-binding cassette transporter G2 single nucleotide polymorphism Y581S on transcellular transport of veterinary drugs using new cell culture models

In commercial dairy production, the risk of drug residues and environmental pollutants in milk from ruminants has become an outstanding problem. One of the main determinants of active drug secretion into milk is the ATP-binding cassette transporter G2/breast cancer resistance protein (ABCG2/BCRP). It is located in several organs associated with drug absorption, metabolism, and excretion, and its expression is highly induced during lactation in the mammary gland of ruminants, mice, and humans. As a consequence, potential contamination of milk could expose suckling infants to xenotoxins. In cows, a SNP for this protein affecting quality and quantity of milk production has been described previously (Y581S). In this study, our main purpose was to determine whether this polymorphism has an effect on transcellular transport of veterinary drugs because this could alter substrate pharmacokinetics and milk residues. We stably expressed the wild-type bovine ABCG2 and the Y581S variant in Madin-Darby canine kidney epithelial cells (MDCKII) and MEF3.8 cell lines generating cell models in which the functionality of the bovine transporter could be addressed. Functional studies confirmed the greater functional activity in mitoxantrone accumulation assays for the Y581S variant with a greater relative V(MAX) value (P = 0.040) and showed for the first time that the Y581S variant presents greater transcellular transport of the model ABCG2 substrate nitrofurantoin (P = 0.024) and of 3 veterinary antibiotics, the fluoroquinolone agents enrofloxacin (P = 0.035), danofloxacin (P = 0.001), and difloxacin (P = 0.008), identified as new substrates of the bovine ABCG2. In addition, the inhibitory effect of the macrocyclic lactone ivermectin on the activity of wild-type bovine ABCG2 and the Y581S variant was also confirmed, showing a greater inhibitory potency on the wild-type protein at all the concentrations tested (5 μM, P = 0.017; 10 μM, P = 0.001; 25 μM, P = 0.008; and 50 μM, P = 0.003). Differential transport activity depending on the genotype together with the differential inhibition pattern might have clinical consequences, including changes in substrate pharmacokinetics (and subsequently pharmacodynamics) and more specifically, changes in secretion of ABCG2 substrates into milk, potentially implying important consequences to veterinary therapeutics.     

Effect of LEPR,ABCG2 and SCD1 Gene Polymorphisms on Reproductive Traits in the Iranian Holstein Cattle

During the last decades, genetic selection for milk production traits has led to increased fertility and health problems in dairy cattle. The aim of this study was to investigate the impact of three polymorphisms located in the ATP‐binding cassette superfamily G member 2 transporter (ABCG2), stearoyl‐CoA desaturase 1 (SCD1) and leptin receptor (LEPR) genes on reproductive traits and somatic cell count (SCC). The analysis was conducted on 408 randomly selected cows. The SNPs within the genes (LEPR, ABCG2 and SCD1) were genotyped using the PCR‐RFLP method. All three possible genotypes were observed for SCD1‐T878C and LEPR‐T945M SNPs, but not for ABCG2‐Y581S SNP. LEPR‐T945M and ABCG2‐Y581S SNPs had no statistically significant effect on the studied reproductive traits and SCC. However, SCD1‐T878C SNP were negatively and significantly related to pregnancy length, dry days and open days (p < 0.05), which lead to decreased profitability in dairy herds. The results suggest that the T878C SNP of SCD1 might be useful as a DNA marker to decrease reproductive problems and improve production traits in Iranian Holstein dairy cows.     

牛乳中黄曲霉毒素B_1和M_1检测方法的比较

比较了免疫亲和柱-高效液相色谱法(HPLC)和酶联免疫吸附法(ELISA)测定牛乳中黄曲霉毒素B1(AFB1)和M1(AFM1)的回收率、方法检出限及精密度。结果表明,HPLC法测定AFB1、AFM1的检测限分别为0.02和0.01μg·L-1,ELISA法测定AFB1、AFM1的最低检出限分别为0.05和0.02μg·L-1;阴性牛乳试样AFB1(0.003、0.006、0.012、0.024μg·kg~(-1))和AFM1(0.005、0.01、0.025、0.05μg·kg~(-1))的回收率试验表明,HPLC法测定AFB1与AFM1的回收率分别为77.58%~82.81%和77.51%~82.23%,变异系数分别为1.93%和3.51%;ELISA法测定AFB1与AFM1回收率分别为77.28%~79.11%和75.40%~76.34%,变异系数分别为2.20%和3.80%,两种方法测定AFB1的回收率差异不显著(P0.05),当AFM1添加浓度大于0.025μg·kg~(-1)时,HPLC法回收率显著高于ELISA法(P0.05);综上,两种方法灵敏度高、重复性好,HPLC法测定高浓度AFM1时准确性优于ELISA法。     

Milk secretion of nitrofurantoin,as a specific BCRP/ABCG2 substrate,in assaf sheep: modulation by isoflavones1

Studies on residues in milk used for human consumption have increased due to health concerns and priority interest in the control of potentially risky drugs. The protein BCRP/ABCG2, present in the mammary epithelia, actively extrudes drugs into milk and can be modulated by isoflavones. Nitrofurantoin is a specific BCRP substrate which is actively excreted into milk by this transporter. In this research, we studied nitrofurantoin transport into milk in four experimental groups: G1‐calves fed forage with isoflavones; G2‐calves fed forage with isoflavones and administered exogenous genistein and daidzein; G3‐calves fed forage without isoflavones; G4‐calves fed forage without isoflavones and administered exogenous genistein and daidzein. Results show increased levels of nitrofurantoin in milk from calves without isoflavones (G3) and decreased nitrofurantoin residues in milk when isoflavones were present, either by forage (G1 and G2) or by exogenous administration (G4). The values of C_max in milk were significantly lower in those groups with isoflavones in forage (G1, G2). Plasma levels were low and unmodified among the groups. Inter‐individual variation was high. All these results seem to point to a feasible control of drug secretion into milk through isoflavones in the diet when the drug is a good BCRP/ABCG2 substrate.     

Comparison between stearoyl‐CoA desaturase expression in milk somatic cells and in mammary tissue of lactating dairy cows

Stearoyl‐CoA desaturase (SCD) is an important enzyme in the bovine mammary gland, where it inserts a cis‐double bond at the Δ9 position in a wide range of fatty acids. Investigating SCD expression in the bovine mammary gland generally requires invasive biopsy to obtain mammary tissue. The aim of this study was to evaluate the use of milk somatic cells as a non‐invasive alternative to biopsy for measuring mammary SCD expression in dairy cows. Both milk somatic cells and mammary tissue were collected from 14 Holstein‐Friesian cows and used for analysis of SCD expression by real‐time PCR. The SCD5 mRNA levels in mammary tissue compared with SCD1 were low, and for several milk somatic cell samples, SCD5 expression was even below the limit of detection. A significant relationship was found between SCD1 expression in milk somatic cells and in mammary tissue. In addition, SCD1 expression in milk somatic cells was significantly related to Δ9‐desaturase indices in milk, which are commonly used as an indicator of SCD1 activity within the mammary gland. Our study showed that milk somatic cells can be used as a source of mRNA to study SCD1 expression in dairy cows, offering a non‐invasive alternative to mammary tissue samples obtained by biopsy.     

奶牛养殖业中黄曲霉素污染和预防控制

奶牛摄入含有较高黄曲霉菌及毒素的饲料,奶牛代谢功能受到损害,抑制免疫机能,使得乳房炎增加,产奶量下降及奶牛体质下降。黄曲霉毒素B1在奶牛体内转换为黄曲霉毒素M1,并分布在牛奶中,人食用含有超标黄曲霉毒素Ml的牛奶后,能使人发生肝炎、肝癌[1]。作者从事奶牛科学养殖技术服务工作,为了预防和控制奶牛场黄曲霉毒素的危害,提供有关黄曲霉素危害、预防控制措施,有一定的利用性。     

The livestock photosensitizer, phytoporphyrin (phylloerythrin), is a substrate of the ATP-binding cassette transporter ABCG2

Hepatogenous photosensitization occurs in livestock following damage to the liver or biliary apparatus that results in impaired excretion of phytoporphyrin (phylloerythrin), a photosensitizer. Based on earlier observations that porphyrin-based photosensitizers are substrates of the ATP-binding cassette transporter ABCG2, we examined the ability of the hepatic transporters ABCB1 (P-glycoprotein) and ABCG2 to transport phytoporphyrin. Transport of phytoporphyrin was blocked by the ABCG2-specific inhibitor fumitremorgin C (FTC) in human embryonic kidney cells transfected with full length human ABCG2, while no transport by cells transfected with human ABCB1 was noted. FTC-inhibited transport of phytoporphyrin was also demonstrated in ABCG2-expressing LLC-PK1 pig kidney cells, consistent with the idea that the pig orthologue, like human ABCG2, transports the photosensitizer. ABCG2 expression was confirmed by immunohistochemistry in the hepatocytes of cow, pig and sheep livers. We conclude that phytoporphyrin is a substrate for ABCG2 and that the transporter is likely responsible for its biliary excretion.     

ABCG2 transporter: therapeutic and physiologic implications in veterinary species

Drug transporters significantly influence drug pharmacokinetics and pharmacodynamics. While P-glycoprotein, the product of the MDR1 (ABCB1) gene, is the most well-characterized ABC transporter, the pharmacological importance of a related transporter, ABCG2, is starting to be realized in veterinary medicine. Based primarily on human and rodent studies, a number of clinically relevant, structurally and functionally unrelated drugs are substrates for ABCG2. ABCG2 is expressed by a variety of normal tissues including the intestines, renal tubular cells, brain and retinal capillary endothelial cells, biliary canalicular cells, and others, where it functions to actively extrude substrate drugs. In this capacity, ABCG2 limits oral absorption of substrate drugs and restricts their distribution to privileged sites such as the brain and retina. ABCG2 is also expressed by tumor cells where it functions to limit the intracellular accumulation of cytotoxic agents, contributing to multidrug resistance. Several ABCG2 polymorphisms have been described in human patients, some of which result in altered drug disposition, increasing susceptibility to adverse drug reactions. Additionally, ABCG2 polymorphisms in humans have been associated with disease states such as gout. Feline ABCG2 has recently been demonstrated to have several amino acid differences at conserved sites compared with 10 other mammalian species. These amino acid differences adversely affect transport function of feline ABCG2 relative to that of human ABCG2. Furthermore, these differences appear to be responsible for fluoroquinolone-induced retinal toxicity in cats and may play a role in acetaminophen toxicity as well. Studies in rodents and sheep have determined that ABCG2 expressed in mammary tissue is responsible for the secretion of many compounds (both therapeutic and toxic) into milk. Finally, data in rodent models suggest that ABCG2 may play an important role in regulating a number of physiologic pathways involved in protecting erythrocytes from oxidative damage.     

Effects of phenylalanine and threonine oligopeptides on milk protein synthesis in cultured bovine mammary epithelial cells

This study was conducted to investigate the effects of phenylalanine (Phe) and threonine (Thr) oligopeptides on α_s1 casein gene expression and milk protein synthesis in bovine mammary epithelial cells. Primary mammary epithelial cells were obtained from Holstein dairy cows and incubated in Dulbecco's modified Eagle's medium‐F12 medium (DMEM/F12) containing lactogenic hormones (prolactin and glucocorticoids). Free Phe (117 μg/ml) was substituted partly with peptide‐bound Phe (phenylalanylphenylalanine, phenylalanyl threonine, threonyl‐phenylalanyl‐phenylalanine) in the experimental media. After incubation with experimental medium, cells were collected for gene expression analysis and medium was collected for milk protein or amino acid determination. The results showed that peptide‐bound Phe at 10% (11.7 μg/ml) significantly enhanced α_s1 casein gene expression and milk protein synthesis as compared with equivalent amount of free Phe. When 10% Phe was replaced by phenylalanylphenylalanine, the disappearance of most essential amino acids increased significantly, and gene expression of peptide transporter 2 and some amino acid transporters was significantly enhanced. These results indicate that the Phe and Thr oligopeptides are important for milk protein synthesis, and peptide‐bound amino acids could be utilised more efficiently in milk protein synthesis than the equivalent amount of free amino acids.     

In vitro effects of very low levels of aflatoxin B₁ on free radicals production and bactericidal activity of bovine blood neutrophils

As one of the most potent and hazardous feed/food-originated mycotoxins, aflatoxin (AF) B? is regarded as a potent immunosuppressor in dairy cows. Neutrophils (PMN), as key effector cells against pathogens, have a high potential to kill engulfed microbes. To investigate the in vitro effects of very low doses of AFB? on blood PMN functions, we examined the effects of biologically relevant concentrations of AFB? on the phagocytosis and non-phagocytosis dependent luminol, representative of mainly intracellular free radicals, and isoluminol, representative of mainly extracellular free radicals, chemiluminescence (CL), necrosis and apoptosis of PMN. Isolated blood PMN from healthy dairy cows (n=12) were exposed to 0, 0.01, 0.05 and 0.5 ng/ml of AFB? for 0.5 and 18 h depending on the assay. Further, blood PMN of healthy dairy cows (n=8) were exposed to 0.5 ng/ml of AFB? for 3h and myeloperoxidase (MPO) activity, superoxide anion (O??) production, phagocytosis and killing activities against Staphylococcus (S.) aureus and Escherichia (E.) coli, were examined. Though the effect of extremely low doses of AFB? were less pronounced, at 0.5 ng/ml the production of free radicals was greatly enhanced, especially extracellularly. In contrast to isoluminol CL, the AFB?-treated PMN showed a remarkably impaired phagocytosis-depended luminol CL. PMN necrosis and apoptosis were not affected by AFB?. MPO activity, O?? production, phagocytosis rates and killing of E. coli and S. aureus by AFB?-treated PMN were significantly lower than those of non-treated ones. Our results show the extracellularly pro-oxidant and antiphagocytic properties of very low doses of AFB? for bovine PMN. The scope of the suppressive effects of the in vitro AFB? levels on cellular innate immune functions should be considered for high yielding dairy cows.     

复合酵母培养物对奶牛产奶性能、氮排放及血液生化指标的影响

为研究饲料中添加复合酵母培养物对奶牛产奶性能、氮排放及血液生化指标的影响,选取年龄、体重、产奶量及泌乳期相近(135±15) d的荷斯坦奶牛24头,随机分为4组,每个处理6个重复,对照组和3个试验组的复合酵母培养物添加量分别为精料浓度的0,0.8%,1.0%,1.2%,随精料饲喂,测定产奶量、乳成分、氮排放及血液生化指标,结果表明,1)试验2组日均产奶量显著高于对照组(P<0.05),各试验组分别比对照组提高8.48%,10.05%,8.97%。2)复合酵母培养物能显著提高乳脂和乳蛋白率(P<0.05),显著降低牛奶体细胞数(P<0.05),以试验2组最低。3)在氮排放量上,试验2组显著低于对照组(P<0.05),各试验组比对照组分别降低8.47%,12.01%,9.36%。4)在血液生化指标方面,复合酵母培养物能提高血清中总蛋白、球蛋白、血糖、胰岛素水平(P<0.05),降低尿素氮水平(P<0.05)。由此可见,本试验条件下,综合考虑产奶量、乳成分、氮排放及血液生化指标,复合酵母培养物的最佳添加量为精料浓度的1.0%。     

乳及乳制品中黄曲霉毒素M_1研究进展

随着我国经济的发展与人们生活水平的提高,具有丰富营养价值的乳及乳制品逐渐成为人们日常生活的必需消费品。与此同时,人们对乳及乳制品的期待不再局限于量的提高,而是对其质量和安全提出了更高的要求。由于2011年发生的牛奶中黄曲霉毒素M_1(AFM_1)含量超标事件,以及相比于成人,婴幼儿更易受到牛奶中AFM_1的损害,人们开始更加重视乳及乳制品中存在的AFM_1污染问题。本文在国内外已有文献报道基础上,对乳及乳制品中AFM_1的来源与生物学性质、污染与限量标准、检测与防控技术进行综述。     

Milk production,nutrient digestibility and nitrogen balance in lactating cows fed total mixed ration silages containing steam‐flaked brown rice as substitute for steam‐flaked corn,and wet food by‐products

The objective of this study was to evaluate the effect of substituting brown rice grain for corn grain in total mixed ration (TMR) silage containing food by‐products on the milk production, whole‐tract nutrient digestibility and nitrogen balance in dairy cows. Six multiparous Holstein cows were used in a crossover design with two dietary treatments: a diet containing 30.9% steam‐flaked corn (corn TMR) or 30.9% steam‐flaked brown rice (rice TMR) with wet soybean curd residue and wet soy sauce cake. Dietary treatment did not affect the dry matter intake, milk yield and compositions in dairy cows. The dry matter and starch digestibility were higher, and the neutral detergent fiber digestibility was lower for rice TMR than for corn TMR. The urinary nitrogen (N) excretion as a proportion of the N intake was lower for rice TMR than for corn TMR with no dietary effect on N secretion in milk and fecal N excretion. These results indicated that the replacement of corn with brown rice in TMR silage relatively reduced urinary N loss without adverse effects on feed intake and milk production, when food by‐products such as soybean curd residue were included in the TMR silage as dietary crude protein sources.     

Effect of using Acacia mearnsii tannin extract as a feed additive on nutritional variables and productive performance in dairy cows grazing a temperate pasture

This study was carried out to evaluate the impact of including Acacia mearnsii tannin extract (TA) as a feed additive on nutrition and productive performance of dairy cows grazing a high‐quality temperate pasture and receiving supplementation with a concentrate feedstuff. Fourteen multiparous Holstein cows were assigned to either of the following treatments: concentrate without or with 20 g TA/kg dry matter (DM). Concentrate intake accounted for 32% of the total DM intake. Tannin addition increased the herbage DM intake by 22% (p < .05). There was no effect of TA inclusion on milk yield, milk composition, milk nitrogen (N) excretion, milk and plasma urea‐N concentration, urinary excretion of total N, urea‐N, and purine derivatives. However, TA inclusion increased the N intake and retention, total N excretion in manure, fecal N to urine N ratio, and decreased the dietary N efficiency for milk production and the percentage of ingested N excreted in urine (p < .05). In conclusion, supplementing dairy cows grazing a high‐quality temperate pasture with a concentrate containing 20 g TA/kg DM showed the potential of decreasing the proportion of ingested N excreted in urine without affecting the productive performance.     

Proteomic analysis to unravel the effect of heat stress on gene expression and milk synthesis in bovine mammary epithelial cells

Heat stress can play a negative effect on milk yield and composition of dairy cattle, leading to immeasurable economic loss. The basic components of the mammary gland are the alveoli; these alveolar mammary epithelial cells reflect the milk producing ability of dairy cows. In this study, we exposed bovine mammary epithelial cells to heat stress and compared them to a control group using isobaric tags for relative and absolute quantitation combined with liquid chromatography coupled with tandem mass spectrometry. Compared with a control group, 104 differentially elevated proteins (>1.3‐fold) and 167 decreased proteins (<0.77‐fold) were identified in the heat treatment group. Gene Ontology analysis identified a majority of the differentially expressed proteins are associated in cell‐substrate junction assembly, catabolic processes and metabolic processes. Some of these significantly regulated proteins were related to the synthesis and secretion of milk, such as milk protein and fat. This finding was further supported by the results obtained from the reduced β‐casein expression through the system of plasminogen activator – plasminogen – plasmin and decreased fatty acid synthase could partly explain why milk fat synthesis ability of dairy cows decreased under heat stress. Our results highlight the effects of heat stress on synthesis of milk protein and fat, thus providing additional clues for further studies of heat stress on dairy milk production.