{'fraction_non_alphanumeric': 0.033824157063593686, 'fraction_numerical': 0.01899274434485702, 'mean_word_length': 4.725717776420281, 'pattern_counts': {'":': 0, '<': 2, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
De novo transgenerational inheritance of male rat hyperactivity by rotenone

— There is growing evidence of transgenerational effects of a single exposure to chemicals, whose mechanism is implicated to be epigenetic. However, it is largely unknown whether psychiatric dis eases such as ADHD or autism caused by environmental chemicals might be transmitted. Rotenone (3 mg/kg), a dopaminergic toxin was orally exposed to Wistar male pups at 5-day old. Their spontaneous motor activity was higher 1.3 fold than that of control rats at 11 weeks of age. At 26 weeks of age, the hyperac tive rat (F0) was mated with Wistar female rats. We established the two strains of such mating and found the spontaneous motor activity of the offspring (F1) were much higher 1.5~2.0 fold than those of both control offspring and the parents. Thus, in this study I show the rat hyperactivity caused by neonatal rote none lesions was transmitted to next generation, indicating the de novo inheritance.


INTRODUCTION
Attention deficit hyperactivity disorder (ADHD) is characterized by behavioral and cognitive symptoms such as hyperactivity, inattention, disorganization, and impulsivity (Goldman et al., 1998;Gozal and Molfese, 2005;Gordon and Mitchell 2009;Rappley, 2005). The etiology is considered to be multifactorial. Interaction of genetic and environmental risk factors would be affected in expression of the disorder. Genetic studies have shown the association of 7-repeat alleles of D4 receptor gene with the occurrence of ADHD (Franke et al., 2012;LaHoste et al., 1996). Many environmental factors seem to cluster around pregnancy and birth, including maternal smoking, alcohol consumption and stress during pregnancy.
It was shown that vinclozolin, an endocrine disruptor exerted the effects on male fertility and that the effects were transmitted to F 4 generation even only when the original gestating mother (F 0 ) of F 1 generation received a transient chemical treatment (Anway et al., 2005). It might be mediated through epigenetic phenomena.
Here, their report allowed us to examine if rat hyperactivity caused by environmental chemical, rotenone toxicity would be transgenerated.

Animals and treatments with chemicals
All animal experiments were carried out in strict accordance with the Experiment and Related Activities in Academic Research Institutions guidelines, under jurisdiction of the Ministry of Education, Culture, Sports, Science and Technology, Japan. The protocol was approved by the Committee on the Ethics of Animal Experiments of the National Institute for Environmental Studies (NIES), Japan. In addition, this study was carried out in compliance with the ARRIVE guidelines. Pregnant Wistar rats were obtained from Clea Japan (Tokyo, Japan). They were maintained in home cages and fed with a standard laboratory chow (MF diet, Oriental Yeast Corp., Tokyo, Japan) and distilled water ad libitum at 22°C on a lightdark cycle (12 hr/12 hr) for at least one week. All animal care procedures were in accordance with NIES guidelines. About 50 male pups were born from 10 pregnant rats and 5-7 pups were randomly housed. The male pups were selected to be 10~14 g body weight at 5 days of age. They were weaned at 3 weeks of age.
Rotenone was suspended in 30% (w/w) milk-oil solution which was composed of nonfat milk (Meiji Co., Tokyo, Japan) and olive oil, and 3 mg/kg of rotenone was orally administered into the pups at 5 days of age. Control rats were administered with vehicle (30 μL) alone.

Mating
Control or the treated male rats (F 0 ) were mated with females from different litters, respectively at 26~30 weeks of age (Fig. 1). The offspring (F 1 ) were obtained, and their spontaneous motor activity was measured by Supermex system as below. Two F1 strains were established, and designated as epi5A and epi5B, respectively.

Measurements of spontaneous motor activity
To examine the behavioral effects of rotenone, we employed the Supermex system (Muromachi Kikai, Tokyo, Japan). A Supermex sensor head consists of paired infrared pyroelectric detectors that measure the radiated body heat of the animal. This system detects any object with a temperature at least 5°C higher than background within a cone-shaped area with a 6 m diameter and a 110°vertex. The sensor monitors motion in multiple zones of the cage through an array of Fresnel lenses placed above the cage and movement of the animal in the X, Y, and Z axis can be covered. A Supermex has the ability to analyze up to 64 channels with an optional instrument, an interface for data recording (DI-064W). Output of the sensor signals representing the magnitude of the rat's movement is transmitted by an interface device to a personal computer and is digitally converted and processed by the CompACT AMS software.
Spontaneous motor activity of the rats was individually measured in a home cage. We measured the activity counted by this system for 15 min intervals for a period of 22 hr. Food and water were fully available ad libitum from the beginning of measurement and rats were never disturbed in any way.

Statistics
Statistical analyses were carried out using the Microsoft Excel 365 software (Tokyo, Japan). Total activity in the nocturnal phase was analyzed by Student's t-test after ANOVA (analysis of variance).

Fig. 1. Illustration of rat generation in this study. Rotenone
(3 mg/kg; top left) was orally administered to male pups at 5 days of age (top), resulting in hyperactive rat (F0). Then, in adulthood, male hyperactive rat was mated with female control rat, resulting in hyperactive rat (F1; bottom). Note that the hyperactive rat (F0) never receives any rotenone.

RESULTS AND DISCUSSION
To examine whether rat hyperactivity elicited by rotenone would be de novo inheritance or not, we fi rst created hyperactive rats (F0) by neonatal rotenone lesion. Rotenone (3 mg/kg) was orally exposed to 5-day old male pups and their spontaneous motor activity was measured. Figure 2A shows that rotenone significantly increased motor activity through the nocturnal phase of the lightdark cycle.
We then examined the transgeneration of rat hyperactivity. Male control or male hyperactive rats (F0) were mated with female control rats from different litters, respectively. The off spring (F1) were obtained. Two F1 strains were established, designated as epi5A and epi5B, respectively. Their spontaneous motor activity was measured by Supermex system. Figure 2B shows that spontaneous motor activity of both F1 rats were signifi cantly higher than that of control rats; they were comparable to that of F0. Figure 2C shows the total spontaneous motor activities in the dark periods (12 hr) of F0 and F1 rats. The extent of hyperactivity in off spring was larger than that of parents (p < 0.05). Spontaneous motor activity in F 0 treated rats was 1.3 times higher than that of control rats, while it was 1.5~2 times in F 1 off spring. Thus, this is not soft inheritance.
The vulnerability of the developing brain is dependent on, particularly the period of exposure to chemicals (Jacobson, 1991;Rice and Barone, 2000). In the 5-day old rats, developmental processes such as diff erentiation and synaptogenesis are incomplete (Vorhees, 1986;Rice and Barone, 2000).
In this study, I demonstrated that a single dose of rotenone (3 mg/kg) was orally administered to male pups at 5 days of age, being hyperactive at adulthood. The male hyperactive rats were then mated with female rat, resulting in the hyperactive generation (F1). The extent of hyperactivity was not decreased. This is strongly indicated the de novo inheritance of rotenone toxicity which was caused at F0 without soft matters. Zhu et al. (2014) demonstrate that mouse hyperactivity, which was generated by mating of a female mouse preexposed to nicotine with a male mouse could be transmitted to offspring via the maternal line. The exposure of pregnant dams to nicotine was kept during pregnancy. It was mediated via a maternal line. However, de novo eff ect has not still been unknown.
Neonatal lesion by an epigenetic modifier, valproic acid could elicit rat hyperactivity (manuscript in preparation). Thus it is possible that rotenone toxicity seen in F0 might be mediated to F1 generation through epigenetic mechanism. This possibility is under investigation.

ACKNOWLEDGMENTS
We thank Mrs Eiko Shimaya and Ruriko Usu for data mining. This work was supported by a grant 17K09180 of KAKENHI. of neonatal rotenone (3 mg/kg)-treated rats ( fi lled circles ) was measured using the Supermex system at adulthood (F0). Measured activity during 2-hr interval is plotted. Control rats were given 30 μL of vehicle alone ( open circles ). Data were represented as a fold of the basal SMA of control rats (ctrl) at diurnal periods. B; Male hyperactive rats were mated with female control rats, resulting in hyperactive rats (F1), whose SMA was represented with that of F0 rats. There are two strains of F1 generation, designated as epi5A ( blue fi lled square ) and epi5B ( blue fi lled circles ). Data were represented as an fold of the basal SMA of control rats ( ctrl ) at diurnal periods. C; SMA during nighttime of all generations was integrated, as indicated. Data were represented as a percentage of those of control rats ( ctrl ), and indicated as mean ± s.e. *Signifi cantly diff erent from control rats ( p < 0.05).