The protective function of taurine on pesticide-induced permanent neurodevelopmental toxicity in juvenile rats.

Numerous studies have confirmed that prenatal or early postnatal exposure to pesticides can lead to functional deficits in the developing brain. This study aimed to investigate whether combined exposure to paraquat (PQ) and maneb (MB) during puberty could cause permanent toxic effects in the neural system of rats. In addition, the neuroprotective function of taurine (T) and its possible mechanism were investigated. Rats were administered PQ + MB intragastrically for 12 continuous weeks, while taurine dissolved in water was fed to the rats for 24 continuous weeks. In the behavioral tests, the rats' trajectories became complex, and the reaction latencies and mistake frequencies increased. Significant changes were found in the hippocampal neurons of the PQ + MB groups but not in the taurine treatment groups. PQ + MB stimulated cAMP to reduce the production of protein kinase A (PKA) and inhibited the activation of other elements, such as brain-derived neurotrophic factor (BDNF), cAMP response element binding protein (CREB), phospho-CREB (p-CREB), immediate-early genes (IEGs)Arc, and c-Fos. Importantly, taurine regulated the level of cAMP and the expression of the abovementioned proteins. Together, our findings implied that adolescent exposure to PQ + MB may impact the behavior and cognitive function of rats via the cAMP-PKA-CREB signaling pathway, while taurine may in turn exert neuroprotection by diminishing these impacts.

In vitro and in vivo impairment of embryo implantation by commonly used fungicide Mancozeb.

The fungicide Mancozeb is an endocrine-disrupting chemical and the mode of action of Mancozeb on embryo implantation is largely unknown. Mancozeb (1 and 3 µg/ml) significantly reduced Jeg-3 trophoblastic spheroids attachment to endometrial epithelial Ishikawa cells. Mancozeb treatment from gestation day (GD) 1 to GD8 or from GD4 to GD8 significantly lowered the number of implantation sites with higher incidence of morphological abnormalities in the reproductive tissues. However, these were not seen in the treatment from GD1 to GD4. Mancozeb at 30 mg/kg BW/d did not alter the expression of p53, COX-2, or PGFS transcripts in the uterus, but down-regulated the PGES transcript and protein. Mancozeb treatment in human endometrial stromal cells did not alter the decidualization response, but the morphological transformation was impaired. Taken together, exposure to Mancozeb affected embryo implantation probably through the modulation of decidualization and to delineate the exact mode of action needs further investigations.

Vitamins E and C prevent apoptosis of testicular and ovarian tissues following mancozeb exposure in the first-generation mouse pups.

The aim of this study was to evaluate the role of apoptosis in the first-generation pups' testicular and ovarian tissue changes following mancozeb (MNZ) administration during intrauterine and lactating periods and also the preventive effect of the co-administration of vitamins E and C on these changes. Naval Medical Research Institute (NMRI) pregnant mice were randomly divided into six groups: control, vehicle, MNZ, vitamin E plus MNZ, vitamin C plus MNZ and vitamins E and C plus MNZ. Administered doses of MNZ and vitamins E and C were 500, 200 and 100 mg/kg of body weight, respectively. These agents were administered to the animals by oral gavage every 2 days. Vitamin treatment was carried out 30 min prior to MNZ administration. Treatment was started on the second day of gestation and continued until weaning. Separated testes and ovaries of animals were prepared for apoptosis detection by terminal deoxynucleotidyl transferase end-labeling (TUNEL) staining. The percentage of TUNEL-positive cells was reported using the 4,6-diamidino-2-phenylindole method. As compared to the control and vehicle groups, MNZ induced a significant increase ( p < 0.001) in the number of TUNEL-positive cells. The administration of both vitamins E and C alone and together significantly ( p < 0.001) prevented the apoptotic impacts of MNZ. The preventive effect of the co-administration of these vitamins on the ovary was greater compared to the single administration of vitamins E ( p < 0.001) or C ( p < 0.001). Meanwhile, the results revealed the stronger preventive effect of vitamin C as compared to E on testicular tissue ( p < 0.05). The apoptotic impact of MNZ exposure during intrauterine and lactating periods on first-generation testicular and ovarian tissues was significant. The co-administration of vitamins E and C could prevent MNZ-induced testicular and ovarian changes.

Silymarin- and melatonin-mediated changes in the expression of selected genes in pesticides-induced Parkinsonism.

Parkinson's disease (PD) is the second most unconcealed neurodegenerative disorder labelled with motor impairments. Two pesticides, manganese ethylene-1,2-bisdithiocarbamate (maneb) and 1,1'-dimethyl-4,4'-bipyridinium dichloride (paraquat), together, are reported to increase the incidence of PD in humans and Parkinsonism in mice. Conversely, silymarin and melatonin, two naturally occurring antioxidants, rescue from maneb- and paraquat-induced Parkinsonism. The study examined silymarin- and melatonin-mediated changes in the expression of selected genes in maneb- and paraquat-induced Parkinsonism employing mouse discover chips microarrays. The mice were treated intraperitoneally (i.p.), daily, with silymarin (40 mg/kg) or melatonin (30 mg/kg) for 9 weeks along with vehicles. Subsets of animals were also treated with maneb (30 mg/kg; i.p.) and paraquat (10 mg/kg; i.p.), twice a week, for 9 weeks. Whilst the expression of genes in the striatum was determined by microarray, the expression of randomly selected transcripts was validated by quantitative real-time polymerase chain reaction (qRT-PCR). Combined maneb- and paraquat-treatment altered the expression of several genes associated with apoptosis, inflammation, cell cycle, cell-signalling, etc. pathways. Silymarin and melatonin significantly resisted the changes in the expression of a few genes related to apoptosis, inflammation, cell cycle, cell-signalling, etc. The expression patterns of seven randomly selected genes were analyzed by qRT-PCR, which were found to follow the similar trends, as observed with microarray. The results obtained from the study thus demonstrate that despite resemblances, silymarin and melatonin differentially offset maneb- and paraquat-induced changes in transcriptome.

Assessment of Mancozeb Exposure, Absorbed Dose, and Oxidative Damage in Greenhouse Farmers.

Mancozeb (MNZ) is a fungicide commonly employed in many countries worldwide. This study assesses MNZ absorption dynamics in 19 greenhouse farmers, specifically following dermal exposure, aiming to verify the efficacy of both preventive actions and protective equipment. For data collection, a multi-assessment approach was used, which included a survey to record study population features. MNZ exposure was assessed through the indirect measurement of ethylene thiourea (ETU), widely employed as an MNZ biomarker. The ETU concentration was measured with the patch method, detecting environmental ETU trapped in filter paper pads, applied both on skin and working clothes, during the 8 h work shift. Urine and serum end-of-shift samples were also collected to measure ETU concentrations and well-known oxidative stress biomarkers, respectively, namely reactive oxygen metabolites (ROMs), advanced oxidation protein products (AOPPs), and biological antioxidant potential (BAP). It was observed that levels of ETU absorbed and ETU excreted were positively correlated. Additionally, working clothes effectively protected workers from MNZ exposure. Moreover, following stratification of the samples based on the specific working duty (i.e., preparation and spreading of MNZ and manipulation of MNZ-treated seedlings), it was found that the spreading group had higher ETU-related risk, despite lower chronic exposure levels. AOPP and ROM serum levels were higher in MNZ-exposed subjects compared with non-exposed controls, whereas BAP levels were significantly lower. Such results support an increase in the oxidative stress upon 8 h MNZ exposure at work. In particular, AOPP levels demonstrated a potential predictive role, as suggested by the contingency analysis results. Overall, this study, although conducted in a small group, confirms that ETU detection in pads, as well as in urine, might enable assessment of the risk associated with MNZ exposure in greenhouse workers. Additionally, the measurement of circulating oxidative stress biomarkers might help to stratify exposed workers based on their sensitivity to MNZ. Pivotally, the combination of both ETU measurement and biological monitoring might represent a novel valuable combined approach for risk assessment in farmhouse workers exposed to pesticides. In the future, these observations will help to implement effective preventive strategies in the workplace for workers at higher risk, including greenhouse farmers who are exposed to pesticides daily, as well as to clarify the occupational exposure levels to ETU.

Exposure of Drosophila melanogaster to Mancozeb Induces Oxidative Damage and Modulates Nrf2 and HSP70/83.

Mancozeb (MZ), a manganese- and zinc-containing ethylene-bis-dithiocarbamate, is a broad-spectrum fungicide. Harmful effects of this fungicide have been reported in nontarget organisms via a not fully understood mechanism. Drosophila melanogaster has provided remarkable contributions for toxicological studies. This work was aimed at evaluating the biochemical targets and implication of oxidative stress in MZ-mediated toxicity in drosophilas. Exposure of flies for fifteen days to MZ at 5 and 10 mg/mL through the diet impaired locomotor performance and induced fly mortality. In parallel, it caused lipid peroxidation and reactive oxygen species (ROS) formation and Mn overload. MZ inhibited superoxide dismutase and inducted catalase and glutathione S-transferase activities. Nitric oxide and reduced glutathione levels were significantly decreased by MZ. Heat shock proteins (HSP70 and HSP83) and Nrf2 mRNA levels were significantly augmented in MZ-exposed flies. Our study reinforced the use of Drosophila melanogaster as a reliable model for the study of biochemical targets of pesticides, and based on our data, MZ induced oxidative damage and Mn accumulation in a concentration-dependent manner. An adaptative cellular state was inducted by the lower concentration of pesticide, possibly contributing to the slighter damage observed.

NFE2L2, PPARGC1α, and pesticides and Parkinson's disease risk and progression.

OBJECTIVE: To investigate three expression-altering NFE2L2 SNPs and four PPARGC1α previously implicated SNPs and pesticides on Parkinson's disease (PD) risk and symptom progression. METHODS: In 472 PD patients and 532 population-based controls, we examined variants and their interactions with maneb and paraquat (MB/PQ) pesticide exposure on PD onset (logistic regression) and progression of motor symptoms and cognitive decline (n = 192; linear repeated measures). RESULTS: NFE2L2 rs6721961 T allele was associated with a reduced risk of PD (OR = 0.70, 95% CI = 0.53, 0.94) and slower cognitive decline (ß = 0.095; p = 0.0004). None of the PPARGC1α SNPs were marginally associated with PD risk. We estimate statistical interactions between MB/PQ and PPARGC1α rs6821591 (interaction p = 0.009) and rs8192678 (interaction p = 0.05), such that those with high exposure and the variant allele were at an increased risk of PD (OR ≥ 1.30, p ≤ 0.05). PPARGC1α rs6821591 was also associated with faster motor symptom progression as measured with the UPDRS-III (ß = 0.234; p = 0.001). CONCLUSION: Our study provides support for the involvement of both NFE2L2 and PPARGC1α in PD susceptibility and progression, marginally and through pathways involving MB/PQ exposure.

Neural repair strategies for Parkinson's disease: insights from primate models.

Nonhuman primate models of Parkinson's disease (PD) have been invaluable to our understanding of the human disease and in the advancement of novel therapies for its treatment. In this review, we attempt to give a brief overview of the animal models of PD currently used, with a more comprehensive focus on the advantages and disadvantages presented by their use in the nonhuman primate. In particular, discussion addresses the 6-hydroxydopamine (6-OHDA), 1-methyl-1,2,3,6-tetrahydopyridine (MPTP), rotenone, paraquat, and maneb parkinsonian models. Additionally, the role of primate PD models in the development of novel therapies, such as trophic factor delivery, grafting, and deep brain stimulation, are described. Finally, the contribution of primate PD models to our understanding of the etiology and pathology of human PD is discussed.

Results from two years of field studies to determine Mancozeb based spray programmes with minimal impact on predatory mites in European vine cultivation.

Mancozeb is a dithiocarbamate fungicide with contact activity against a wide range of economically important fungal diseases. Its multi-site mode of action means that to date there have been no recorded incidences of resistance developing despite many years of use on high risk diseases. One such disease, Grape downy mildew (Plasmopara viticola) has developed resistance to a wide range of important oomycete specific fungicides following their introduction onto the market. The role of Mancozeb either as a mixing or alternation partner in helping to manage these resistance situations remains critically important. Historical use patterns for mancozeb in tree and vine crops involved many applications of product at high use rates. Although this gave excellent disease control, a negative impact on predatory mites was often reported by researchers. This has lead to the development of mancozeb spray programmes in vines and other crops with a much reduced impact on predatory mites. A range of field studies was conducted over two years in France, Germany, Italy, Portugal and Spain where 2, 3 or 4 applications of mancozeb containing products were made per season at different spray timings. In this paper findings from field studies over two years in five different vine growing regions in Europe indicated that two to four applications of mancozeb at 1.6 kg a.i./ha as part of a spray programme caused minimal impact on naturally occurring populations of predatory mites which in turn was compatible with Integrated Pest Management programmes and the conservation of predatory mites.

Effects of prenatal paraquat and mancozeb exposure on amino acid synaptic transmission in developing mouse cerebellar cortex.

The goal of this study was to analyze the effects of prenatal exposure to the pesticides paraquat (PQ) and mancozeb (MZ) on the development of synaptic transmission in mouse cerebellar cortex. Pregnant NMRI mice were treated with either saline, 10 mg/kg PQ, 30 mg/kg MZ or the combination of PQ + MZ, between gestational days 12 (E12) and E20. Variation in the levels of amino acid neurotransmitters was determined by HPLC, between postnatal day 1 (P1) and P30. Motor coordination was assessed by locomotor activity evaluation of control and experimental pups at P14, P21 and P30. Significant reductions in the levels of excitatory neurotransmitters, aspartate and glutamate, were observed in PQ-, MZ- or combined PQ + MZ-exposed pups, with respect to control, during peak periods of excitatory innervation of Purkinje cells: between P2-P5 and P11-P15. However, at P30, lower aspartate contents, in contrast with increased glutamate levels, were detected in all experimental groups. During the first two postnatal weeks, delays in GABA and glycine ontogenesis were observed in PQ- and PQ + MZ-exposed pups, whereas notable decrements in GABA and glycine levels were seen in PQ + MZ-exposed animals. Decreased taurine contents were detected at P3 and P11 in PQ- and PQ + MZ-exposed mice. Pups in different experimental groups all showed hyperactivity at P14 and then exhibited reduced locomotor activity at P30. Taken together, our results indicate that prenatal exposure to either PQ or MZ or the combination of both could alter the chronology and magnitude of synaptic transmission in developing mouse cerebellar cortex.

Evaluation of epidemiologic and animal data associating pesticides with Parkinson's disease.

Exposure to pesticides may be a risk factor for developing Parkinson's disease (PD). To evaluate the evidence regarding this association in the scientific literature, we examined both analytic epidemiologic studies of PD cases in which exposure to pesticides was queried directly and whole-animal studies for PD-like effects after systemic pesticide exposure. Epidemiologic studies were considered according to study quality parameters, and results were found to be mixed and without consistent exposure-response or pesticide-specific patterns. These epidemiologic studies were limited by a lack of detailed and validated pesticide exposure assessment. In animal studies, no pesticide has yet demonstrated the selective set of clinical and pathologic signs that characterize human PD, particularly at levels relevant to human populations. We conclude that the animal and epidemiologic data reviewed do not provide sufficient evidence to support a causal association between pesticide exposure and PD.

Field studies to determine mancozeb based spray programmes with minimal impact on predatory mites in European vine cultivation.

Mancozeb is an ethylene bisdithiocarbamate (EBDC) fungicide with contact activity against a wide range of economically important fungal diseases. Its multi-site mode of action means that to date there have been no recorded incidences of resistance developing despite many years of use on high risk diseases. One such disease, Grape downy mildew (Plasmopara viticola) has developed resistance to a number of important oomycete specific fungicides following their introduction onto the market. The role of Mancozeb either as a mixing or alternation partner in helping to manage these resistance situations remains critically important. Historical use patterns for mancozeb in tree and vine crops involved many applications of product at high use rates. Although this gave excellent disease control, a negative impact on predatory mites has been reported by researchers. This has lead to the development of mancozeb spray programmes in vines and other crops with a much reduced impact on predatory mites. A range of field studies was conducted in France, Germany, Italy, Portugal and Spain where either 2 or 4 applications of mancozeb containing products were made per season at different spray timings. These trials covered the representative range of uses, agronomic practices, mite species and geographical locations in Europe. In this paper findings from ten field studies in five different vine growing regions in Europe indicated that two to four applications of mancozeb at 1.6 kg a.i./ha as part of a spray programme caused minimal impact on naturally occurring populations of predatory mites which in turn was compatible with Integrated Pest Management programmes and the conservation of predatory mites.

Chronic exposure to the fungicide maneb may produce symptoms and signs of CNS manganese intoxication.

Manganese (Mn) poisoning, a well-known hazard in miners and industrial workers, shares many features with Parkinson's disease. Two young agricultural workers with a parkinsonian syndrome, who mentioned exposure to the fungicide maneb (manganese ethylene-bis-dithiocarbamate), led us to investigate a new possible source of Mn intoxication. Fifty male rural workers with occupational exposure to maneb were compared with 19 rural workers without fungicide exposure. We noted significantly higher prevalence of plastic rigidity with cogwheel phenomenon, headache, fatigue, nervousness, memory complaints, and sleepiness in the exposed group. In addition, we saw other neurologic signs, such as postural tremor, cerebellar signs, and bradykinesia, although without statistical significance. The data suggest that occupational exposure to pesticides containing Mn is a possible source of Mn intoxication of the CNS.

Exposure of nonmigratory pigeons to mancozeb: a sentinel model for humans.

The use of pesticides in agriculture has increased over the last decade. Their widespread, often uncontrolled use causes thousands of people to be daily exposed to environmental agricultural chemicals, resulting in acute and chronic health effects. At present there is a paucity of data on the potential adverse effects of exposure to low levels of mancozeb for prolonged periods. In order to investigate the effects of mancozeb exposure on pulmonary wildlife populations, tracheas and lungs of nine 1- to 7-yr-old nonmigratory pigeons raised near peach orchards and vineyards repeatedly sprayed with the fungicide were examined. The experimental situation allowed us to evaluate the long-term natural toxicity of mancozeb as a sentinel for human populations occupationally exposed to fungicides. The use of nonmigratory pigeons may serve as an important biological source from which helpful data may be obtained for assessing risks to human health and gaining new insight into pathogenetic mechanisms.

Parkinsonism after chronic exposure to the fungicide maneb (manganese ethylene-bis-dithiocarbamate).

Permanent parkinsonism was observed in a man with chronic exposure to the fungicide maneb (manganese ethylene-bis-dithiocarbamate). Symptoms developed at 37 years of age, two years after exposure had ceased. To our knowledge, this is the second report on parkinsonism associated with exposure to maneb. Manganese is a well-known parkinsonigen toxin in humans. More recently, it has been shown that dithiocarbamates can also induce extrapyramidal syndromes. The biochemical effects of manganese and dithiocarbamates are reviewed and their possible neurotoxic mechanisms are discussed. Both of these components may have played a role in this case.

Ethylenethiourea in air and in urine as an indicator of exposure to ethylenebisdithiocarbamate fungicides.

Ethylenethiourea (ETU) is a ubiquitous impurity of the ethylenebisdithiocarbamate (EBDC) fungicides widely used in agriculture and forestry. In the present study, ETU was used as a measure of the exposure to EBDC on potato farms and in pine nurseries during the application of EBDC fungicides and the weeding of the sprayed vegetation. Biological and hygienic monitoring was carried out through the analysis of ETU in the breathing zone and the urine of exposed workers. Even if the concentrations of ETU in the ambient air of pine nurseries exceeded those of potato farms, the concentrations of ETU in the urine of potato farmers exceeded those of pine nursery workers. This result may have been due better protective equipment in the pine nurseries. The excretion rate was 6-10 ng/h during the first 60 h after the cessation of exposure, and it diminished thereafter to 0.2 ng/h over a 22-d observation period.

Effects of fungicides on thyroid function, metabolism, and thermoregulation in cotton rats.

Among the myriad of recent studies on endocrine-disrupting chemicals, relatively few involve thyroid disruption, and most of these address exposure/disruption during embryonic life. Of those involving adult vertebrates, the endpoints examined are thyroid measurements. Even though thyroid disruption could potentially interfere with energy metabolism and thermoregulation such that over-winter survival might be compromised, the possible energetic consequences of these thyroid perturbations have not been investigated. We assessed thyroid function and measured resting metabolic rates of cotton rats chronically exposed to the fungicides vinclozolin or mancozeb. In addition, we measured norepinephrine-induced nonshivering thermogenesis and cold-induced thermogenesis and then cold-acclimated the mancozeb animals. Although thyroid hormone concentrations generally decreased, this was compensated for by an increase in thyroxine turnover (vinclozolin study only) such that thyroxine utilization rate was not different. In addition, there was no difference between the treated and control animals in any of the metabolic parameters measured. It is concluded that wild rodents exposed to these compounds are not energetically compromised.

Anti-implantation effect of a carbamate fungicide mancozeb in albino mice.

Mancozeb, an organocarbamate fungicide, was administered to examine the effect on implantation at doses of 18, 24, 30 and 36 mg/kg body weight/d to normal virgin swiss albino mice for 8 days to pregnant mice. The vaginal smear and body weight of the mice were recorded daily and mice were sacrificed on 9th day of pregnancy. There was a complete inhibition of implantation in 36 mg mancozeb treated mice with 100% pre-implantation loss. There was a partial inhibition of implantation in 24 and 30 mg mancozeb treated mice with 53.44 and 90.16% pre-implantation loss respectively. However, implantation was not affected in 18 mg mancozeb treated mice with 4.92% pre-implantation loss when compared to oil treated controls. To study the temporal effect, the effective dose of 36 mg/kg body weight/d mancozeb was administered orally for 3 and 5 days and on day 3 only. There was a complete inhibition of implantation in 5 days treated mice with 100% pre-implantation loss and partial inhibition of implantation of 3 days treated mice with 75% pre-implantation loss. However, implantation was not affected in mice treated on day 3 only with 1.63% pre-implantation loss when compared to control mice. There was a significant decrease in the diestrus phase with the result there was a concomitant increase in the estrus phase and there was a significant decrease in the uterus weight with 24, 30 and 36 mg and for 3 and 5 days with 36 mg mancozeb treatment. Inhibition of implantation by mancozeb may be due to hormonal imbalance or its toxic effects.

Induction of gonadal toxicity to female rats after chronic exposure to mancozeb.

Mancozeb, a fungicide of ethylenebisdithiocarbamate group was orally administered at doses of 500, 600, 700 and 800 mg/kg body weight/day to normal virgin rats of Wistar strain for 30 days. The vaginal smear and body weight of the rats were recorded daily and rats were sacrificed on 31st day. Estrous cycle was effected by showing a significant decrease in the number of estrous cycle, duration of proestrus, estrus and metestrus with concomitant significant increase in the duration of diestrus in all the mancozeb treated groups when compared with controls. There were a significant decrease in the number of healthy follicles and a significant increase in the number of atretic follicles in all the mancozeb treated groups when compared with controls. The histologic observation of the ovary revealed the presence of less number of corpora lutea and the size of the ovary was also reduced in high doses of mancozeb treated rats. There was a significant increase in the thyroid weight in all the mancozeb treated rats except in 500 mg/kg/d. In rats treated with 500 mg/kg/d showed a significant increase in the level of total lipids in the liver. In rats treated with 600 mg/kg/d mancozeb showed a significant decrease in the levels of glycogen and total lipids in the uterus and total lipids in the liver. In rats treated with 700 mg/kg/d showed a significant decrease in the levels of protein in ovary, glycogen, total lipids, phospholipids and neutral lipids in the uterus and a significant increase in the levels of phospholipids, neutral lipids in the ovary and total lipids, phospholipids and neutral lipids in the liver. In rats treated with 800 mg/kg/d showed a significant decrease in the levels of protein and glycogen in the ovary and protein, glycogen, total lipids, phospholipids and neutral lipids in the uterus and a significant increase in the levels of total lipids, phospholipids and neutral lipids in the ovary and liver when compared with controls. These observed effect of mancozeb on the estrous cycle, follicles and biochemical constituents may be due to imbalance in the hormone or toxic effect.

Effect of chronic mixed pesticide exposure on peripheral and autonomic nerve function.

In a cross-sectional study involving 131 flower bulb farmers (mean age = 43 y) and 67 well-matched controls, peripheral and autonomic nerve functions were examined. The study group had been exposed during a period of 20 y (standard deviation = 7) and applied a similar pesticide package. Lifetime cumulative exposure was estimated based on exposure levels for specific application methods and duration of exposure. Exposure-related decreased conduction velocities were found in the motor fibers of the median (-1.1 m/s) and peroneal (fast fibers: -1.2 m/s, slow fibers: -1.3 m/s) nerves, and in the sensory fibers of the median (-1.4 m/s) and sural (-0.9 m/s) nerves. In addition, the refractory period was determined and found to be increased in the sural and peroneal nerves. With regard to the autonomic nerve function, a decrease was found in resting sinus arrhythmia (-10%).