Search

5-Methylcytosine is one of the major epigenetic marks of DNA in living organisms. Some bacterial species possess DNA methyltransferases that modify cytosines on both strands to produce fully-methylated sites or on either strand to produce hemi-methylated sites. In this study, we characterized a DNA methyltransferase that produces two sequences with different methylation patterns: one methylated on both strands and another on one strand. M.BatI is the orphan DNA methyltransferase of Bacillus anthracis coded in one of the prophages on the chromosome. Analysis of M.BatI modified DNA by bisulfite sequencing revealed that the enzyme methylates the first cytosine in sequences of 5ʹ-GCAGC-3ʹ, 5ʹ-GCTGC-3ʹ, and 5ʹ-GCGGC-3ʹ, but not of 5ʹ-GCCGC-3ʹ. This resulted in the production of fully-methylated 5ʹ-GCWGC-3ʹ and hemi-methylated 5ʹ-GCSGC-3ʹ. M.BatI also showed toxicity when expressed in E. coli, which was caused by a mechanism other than DNA modification activity. Homologs of M.BatI were found in other Bacillus species on different prophage like regions, suggesting the spread of the gene by several different phages. The discovery of the DNA methyltransferase with unique modification target specificity suggested unrevealed diversity of target sequences of bacterial cytosine DNA methyltransferase. [ABSTRACT FROM AUTHOR]

Neonicotinoid insecticides (NNIs) are now popular in many agricultural systems across Africa; however, the extent of human exposures to NNIs in African countries is scarcely reported. The present study evaluates neonicotinoid exposures in the consumer population of Kumasi, a cosmopolitan city in Ghana. A total of 75 human urine samples were collected from healthy volunteers (nonfarmers, aged 13–80 yr) and analyzed with a liquid chromatography electrospray ionization tandem mass spectrometry system. Seven NNIs and 3 NNI metabolites were detected in the following pattern (frequency, median concentration, maximum concentration): N‐dm‐acetamiprid (94.7%, 0.41 µg/L, 8.79 µg/L) > imidacloprid (70.7%, 0.15 µg/L, 211.62 µg/L) > N‐(6‐chloro‐3‐pyridylmethyl)‐N‐ethyl‐N′‐methylformamidine (62.2%, 0.43 µg/L, 53.85 µg/L) > 2‐[N‐(6‐chloro‐3‐pyridylmethyl)‐N‐ethylamino]‐2‐(methylimino)acetic acid (56.8%, 0.10 µg/L, 3.53 µg/L) > clothianidin (40%, >limit of quantification [LOQ], 0.45 µg/L) > nitenpyram (18.7%, >LOQ, 0.14 µg/L) ≈ thiamethoxam (18.7%, >LOQ, 0.21 µg/L) > dinotefuran (12.0%, >LOQ, 1.01 µg/L) > acetamiprid (2.7%, >LOQ, 0.08 µg/L) ≈ thiacloprid (2.7%, >LOQ, 0.14 µg/L). Approximately 92% of the subjects were found to be exposed to multiple neonicotinoids simultaneously. The mean, median, and maximum imidacloprid equivalent of the relative potency factor of NNIs were found to be 1.6, 0.5, and 22.52, respectively. The median estimated daily intakes of acetamiprid, imidacloprid, and nitenpyram were 0.47, 1.27, and 0.02 µg/kg/d for females and 0.91, 0.66, and 0.08 µg/kg/d for males, respectively. The maximum daily intakes of all the NNIs were <1% of their chronic reference doses (cRfDs), except for imidacloprid and thiacloprid which recorded maximum daily intakes corresponding to 17.97 and 8.28% of cRfDs, respectively. To the best of our knowledge, the present study is the first biomonitoring report on neonicotinoid insecticides in Africa. Environ Toxicol Chem 2021;40:2306–2318. © 2021 SETAC [ABSTRACT FROM AUTHOR]

Measurement of urinary metanephrines in spot samples is used for the diagnosis of canine pheochromocytoma (PC). We describe a simple analytical method based on liquid chromatography tandem mass spectrometry (LC-MS/MS) for measuring free metanephrine (MN) and normetanephrine (NMN) in spot urine samples. Using the developed method, we evaluated the stability of urinary free-MN and free-NMN at various storing conditions. In addition, we assessed the feasibility of urinary free-MN and -NMN measurement for diagnosing PC. Urine samples were mixed with stable isotope internal standards and thereafter purified by ultrafiltration. The purified samples were analyzed by LC-MS/MS in the multiple reaction monitoring mode after separation on a multimode octa decyl silyl column. The coefficient of variation of free-MN and -NMN measurement was 7.6% and 5.5%, respectively. The linearity range was 0.5-10 µg/l for both analytes. Degradation was less than 10% for both analytes under any of the storage conditions. The median free-NMN ratio to creatinine of 9 PC dogs (595, range 144--47,961) was significantly higher (P<0.05) than that of 13 dogs with hypercortisolism (125, range 52--224) or 15 healthy dogs (85, range 50--117). The developed method is simple and may not require acidification of spot urine. The results of this preliminary retrospective study suggest that the measurement of urinary free metanephrines is a promising tool for diagnosing canine PC. [ABSTRACT FROM AUTHOR]

Objectives: Neonicotinoid insecticides are widely used systemic pesticides with nicotinic acetylcholine receptor agonist activity that are a concern as environmental pollutants. Neonicotinoids in humans and the environment have been widely reported, but few studies have examined their presence in fetuses and newborns. The objective of this study is to determine exposure to neonicotinoids and metabolites in very low birth weight (VLBW) infants. Methods: An analytical method for seven neonicotinoids and one neonicotinoid metabolite, N-desmethylacetamiprid (DMAP), in human urine using LC-ESI/MS/MS was developed. This method was used for analysis of 57 urine samples collected within 48 hours after birth from VLBW infants of gestational age 23–34 weeks (male/female = 36/21, small for gestational age (SGA)/appropriate gestational age (AGA) = 6/51) who were admitted to the neonatal intensive care unit of Dokkyo Hospital from January 2009 to December 2010. Sixty-five samples collected on postnatal day 14 (M/F = 37/22, SGA/AGA = 7/52) were also analyzed. Results: DMAP, a metabolite of acetamiprid, was detected in 14 urine samples collected at birth (24.6%, median level 0.048 ppb) and in 7 samples collected on postnatal day 14 (11.9%, median level 0.09 ppb). The urinary DMAP detection rate and level were higher in SGA than in AGA infants (both p<0.05). There were no correlations between the DMAP level and infant physique indexes (length, height, and head circumference SD scores). Conclusion: These results provide the first evidence worldwide of neonicotinoid exposure in newborn babies in the early phase after birth. The findings suggest a need to examine potential neurodevelopmental toxicity of neonicotinoids and metabolites in human fetuses. [ABSTRACT FROM AUTHOR]

Neonicotinoid insecticides that have been on the market since 1992 have been used globally including in Japan. Because they are sprayed over forests and agricultural areas, inadvertent toxicity in nontarget insects (especially honey bees) and humans is a matter of public concern. However, information on exposure levels and potential health impacts of neonicotinoids in children living around sprayed areas is scarce. Thus, we determined neonicotinoid exposure levels in children living in communities where thiacloprid was used to control pine wilt disease. A total of 46 children (23 males and 23 females) were recruited for the present study, and informed written consent was obtained from their guardians. Urine specimens were collected before, during, and after insecticide spraying events; and atmospheric particulate matter was also collected. Concentrations of thiacloprid and 6 other neonicotinoid compounds were determined in urine samples and in atmospheric particulate matter specimens using liquid chromatography‐electrospray ionization‐tandem mass spectrometry. In urine specimens, thiacloprid concentrations were <0.13 μg/L and were detectable in approximately 30% of all samples. Concentrations of the other neonicotinoids, N‐dm‐acetamiprid, thiamethoxam, dinotefuran, and clothianidin, were 18.7, 1.92, 72.3, and 6.02 µg/L, respectively. Estimated daily intakes of these neonicotinoids were then calculated from urinary levels; although the estimated daily intakes of the neonicotinoids were lower than current acceptable daily intake values, the children were found to be exposed to multiple neonicotinoids on a daily basis. Environ Toxicol Chem 2019;38:71–79. © 2018 SETAC [ABSTRACT FROM AUTHOR]

To evaluate the metabolic responses in tumour cells exposed to ionizing radiation, oxygen consumption rate (OCR), cellular lipid peroxidation, cellular energy status (intracellular nucleotide pool and ATP production), and mitochondrial reactive oxygen species (ROS), semiquinone (SQ), and iron-sulphur (Fe−S) cluster levels were evaluated in human cervical carcinoma HeLa cells at 12 and 24 h after X-irradiation. LC/MS/MS analysis showed that levels of 8-iso PGF2α and 5-iPF2α-VI, lipid peroxidation products of membrane arachidonic acids, were not altered significantly in X-irradiated cells, although mitochondrial ROS levels and OCR significantly increased in the cells at 24 h after irradiation. LC/UV analysis revealed that intracellular AMP, ADP, and ATP levels increased significantly after X-irradiation, but adenylate energy charge (adenylate energy charge (AEC) = [ATP + 0.5 × ADP]/[ATP + ADP + AMP]) remained unchanged after X-irradiation. In low-temperature electron spin resonance (ESR) spectra of HeLa cells, the presence of mitochondrial SQ at g = 2.004 and Fe-S cluster at g = 1.941 was observed and X-irradiation enhanced the signal intensity of SQ but not of the Fe-S cluster. Furthermore, this radiation-induced increase in SQ signal intensity disappeared on treatment with rotenone, which inhibits electron transfer from Fe-S cluster to SQ in complex I. From these results, it was suggested that an increase in OCR and imbalance in SQ and Fe-S cluster levels, which play a critical role in the mitochondrial electron transport chain (ETC), occur after X-irradiation, resulting in an increase in ATP production and ROS leakage from the activated mitochondrial ETC. [ABSTRACT FROM AUTHOR]

Among wild birds, lead (Pb) exposure caused by ingestion of ammunition is a worldwide problem. We aimed to reveal the behavior and toxic effect of Pb caused by ingesting Pb shots in waterfowl. Four male, eight-week old Muscovy ducks (Cairina moschata) were given three Pb shots (approximately 240 mg in total) orally and then fed for 29 days after exposure, simulating a low-dose Pb exposure in wild waterfowl. During the breeding period, blood samples were collected 10 times, and fecal samples every day. Additionally, 22 fresh tissue and 6 bone samples were obtained from each duck through the dissection. Although there were no gross abnormalities, the maximum blood Pb concentration of each duck ranged from 0.6 to 3.7 mg/L, reaching a threshold concentration indicative of clinical symptoms (>0.5 mg/L). δ-aminolevulinic acid dehydratase declined one day after exposure and remained low throughout the feeding period. Hematocrit also tended to decrease, indicating signs of anemia. The highest Pb accumulation was observed in the bones, followed by the kidneys, intestinal tracts, and liver. High Pb accumulation in the bones, which are known to have a long Pb half-life, suggested that Pb would remain in the body and possibly affect bird health beyond 28 days after exposure. Gene expression analysis showed a significant increase in the expression of the toll-like receptor-3 gene, which is involved in virus discrimination in the liver, suggesting a disruption of the immune system. Microbiota analyses showed a correlation between the blood Pb concentration and the abundances of Lachnospiraceae and Ruminococcaceae , suggesting that Pb affects lipid metabolism. These results provide fundamental data on Pb exposure in wild birds and a new perspective on the damage such exposure causes. [Display omitted] • Pb levels in various organs of low-dose-exposed ducks were revealed. • Blood Pb levels reached thresholds at which clinical poisoning is expected. • Signs of anemia were observed through δ-ALAD activity and Ht. • Differences in TLR3 gene expression suggested immune system disruption by Pb. • Lachnospiraceae and Ruminococcaceae changes suggested lipid metabolism alteration. [ABSTRACT FROM AUTHOR]

[Display omitted] • Impacts of organic dietary interventions on neonicotinoid insecticide (NNIs) exposures were determined. • Detection rates of NNIs in organic diet consumers were lower than their conventional counterparts. • Organic diet consumers showed lower multiple exposures to NNIs than their conventional counterparts. • The cumulative levels of NNIs were significantly lower in the organic diet consumers. • Organic dietary interventions resulted in drastic reductions in NNI daily intake rates. Neonicotinoid insecticides (NNIs) are a popular class of insecticides used in various pest management regimens worldwide. Biomonitoring studies continuously report high exposure rates of NNIs in various human populations across the globe. Yet, there is no validated countermeasure for combating the recent exponential rise in NNI exposure rates observed in human populations. The current study assessed the impacts of organic dietary interventions on NNI exposure rates in a Japanese population. A total of 103 volunteers were recruited into the study. Subjects were either served with Organic diets for 5 and 30 days or conventional diets. A total of 919 repeated urine samples were collected from the participants and then subjected to LC-MS/MS analysis to determine urinary concentrations of 7 NNIs parent compounds and an NNI metabolite. Eight NNIs were detected; with a decreasing detection frequency (%Dfs) pattern; desmethyl-acetamiprid (dm-ACE) (64.96%) > dinotefuran (52.12%), imidacloprid (39.61%) > clothianidin (33.95%) > thiamethoxam (28.51%) > acetamiprid (12.62%) > nitenpyram (5.33%) > thiacloprid (2.83%). Dinotefuran, dm-ACE, and clothianidin recorded the highest concentrations in the subjects. The %Df of NNIs in the 5-days or 30-days organic diet group were lower than those of the conventional diet consumers. The organic diet group showed lower rates of multiple NNI exposures than those of the conventional diet consumers. The mean and median cumulative levels of NNIs (median IMI eq) were significantly lower in the organic diet group than the conventional diet group (p < 0.0001). The estimated daily intakes (EDIs) of NNIs were higher in adults than children, but less than 1% of NNI cRfDs, except for clothianidin, which exhibited a %cRfD of 1.32 in children. Compared to the conventional diet group, the 5- and 30-day organic dietary intervention showed drastic reductions in NNI EDIs. Findings from the present study give credence to organic dietary interventions as potential ameliorative strategies for NNI exposure rates in human populations. [ABSTRACT FROM AUTHOR]

Fipronil is a phenylpyrazole insecticide that is widely used as a pesticide and a veterinary drug, although studies suggest that it could be toxic to mammals. The objectives of this study were to examine the pharmacokinetic profile of fipronil in mice, dogs, and cats, and to evaluate its effects on emotional and cognitive behaviors of dogs and cats using the data obtained from mice. The assessment of in vivo kinetics of fipronil was conducted in mice and dogs. We also performed behavioral tests (elevated plus-maze and Y-maze) and measured the levels of neurotransmitters in mice exposed to fipronil. In addition, the in vitro metabolism of fipronil were evaluated using liver microsomes of rats, mice, dogs, and cats. The results revealed that fipronil is distributed throughout the body (blood, brain, adipose tissue, and liver) of mice after dermal application. It was metabolized to fipronil sulfone primarily in the liver. The data on kinetics show that both fipronil and fipronil sulfone have a longer half-life in dogs and cats than in mice. The behavioral tests indicated that fipronil and fipronil sulfone could affect emotional and cognitive behaviors and alter the levels of neurotransmitters (dopamine in the striatum and serotonin in the hippocampus) in mice. Furthermore, we found that dogs and cats have a low ability to metabolize fipronil than mice and rats. However, further comprehensive studies are needed to determine whether fipronil affects the emotional and cognitive behaviors when administered to dogs and cats. To the best of our knowledge, this is the first study to examine the pharmacokinetic data and verify the effects of fipronil on emotional and cognitive behaviors of dogs and cats using the data obtained from mice. [Display omitted] • Fipronil is distributed throughout the body, including blood, brain, adipose tissue, and liver after dermal application. • Fipronil was metabolized to fipronil sulfone mainly in the liver. • Fipronil and fipronil sulfone remain in the blood for a longer time in dogs than in mice. • Fipronil alters the levels of dopamine and serotonin in the brains of mice and changes emotional and cognitive behaviors. • The metabolic effects of fipronil in dogs and cats are much lower than those of mice and rats. [ABSTRACT FROM AUTHOR]

Background: Hepatocellular carcinoma (HCC) is one of the most common primary liver tumors in humans and dogs. Excessive adrenocortical hormone exposure may cause steroid hepatopathy, which may develop into HCC. In our previous study, hyperadrenocorticism (HAC) was a highly concurrent disease in dogs with HCC. Therefore, this study hypothesized that adrenal steroid alterations might be involved in hepatocarcinogenesis and aimed to specify the relationship between HAC and HCC in dogs., Materials and Methods: This study included 46 dogs brought to the Hokkaido University Veterinary Teaching Hospital between March 2019 and December 2020. Owners gave their signed consent for blood collection on their first visit. A total of 19 steroids (14 steroids and 5 metabolites) in the baseline serum of 15 dogs with HCC, 15 dogs with HAC, and 10 dogs with both diseases were quantitatively measured using the developed liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) method., Results: In each group, 11 steroids were detected higher than 50%. The detection rate of steroid hormones did not significantly differ between the groups ( p > 0.05). Principle component analysis (PCA) showed that the steroid profiles of the three groups were comparable. Median steroid hormone concentrations were not significantly different between the study diseases ( p > 0.05)., Conclusion: The developed LC/MS/MS was useful for measuring steroid hormones. Although it was clear that HAC was concurrent in dogs with HCC, none of the serum steroids was suggested to be involved in HCC., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Oo, Sasaki, Ikenaka, Ichise, Nagata, Yokoyama, Sasaoka, Morishita, Nakamura and Takiguchi.)

We developed an analytical method using an on-line column-switching liquid chromatography with triple quadrupole mass spectrometry (LC/MS/MS) for quantifying multiple steroids in serum. Using the developed method, we evaluated the serum concentration of nine steroids (cortisol, corticosterone, cortisone, 11-deoxycortisol, 21-deoxycortisol, deoxycorticosterone, progesterone, 17α-OH-progesterone and aldosterone) in dogs with hyperadrenocorticism (HAC). Serum was mixed with stable isotope internal standards and thereafter purified by the automated column-switching system. The limit of detection ranged 2-16 pg/ml for nine steroids. In the baseline samples, five steroids (cortisol, corticosterone, cortisone, 11-deoxycortisol, and 17α-OH-progesterone) were detected in all dogs. The concentrations of cortisone, 11-deoxycortisol, and 17α-OH-progesterone in dogs with HAC (n=19) were significantly higher those in dogs without HAC (n=15, P<0.02). After the adrenocorticotropic hormone stimulation test, six steroids (cortisol, corticosterone, cortisone, 11-deoxycortisol, 17α-OH-progesterone, and deoxycorticosterone) were above the limit of quantification in all dogs. Cortisol, corticosterone, cortisone, and deoxycorticosterone concentrations of dogs with HAC were significantly higher than those of dogs without HAC (P<0.02). In addition, 11-deoxycortisol and 17α-OH-progesterone concentration was higher in dogs with HAC than in dogs without HAC (P=0.044 and P=0.048, respectively). The on-line column-switching LC/MS/MS would be feasible for measuring multiple steroids in dog serum. The results suggest that cortisone, 11-deoxycortisol, and 17α-OH-progesterone would be related to HAC. Further studies are warranted to assess the clinical feasibility of steroid profile in dogs with HAC.