Pesticide mix and the hepato-brain axis: role of xenobiotic nuclear receptors – HepatoBrain

We have assembled a multi-modal set of techniques to track possible in vivo pathological modifications triggered by dietary pesticide cocktails and to determine the implication of specific nuclear receptors in mediating some of these effects. We use:
1) Metabolic analyses: body weight, glycemia, insulinemia, lipidemia, glucose and insulin intolerance tests
2) Endocrine disruptions analyses: measurement of testosterone, estradiol and corticosterone levels
3) Models of mice invalidated for a xenobiotic receptor (total and liver-specific)
4) Transcriptomic analyses (liver) and metabolomics (liver and urine)
5) Comprehensive battery of behavioral testing repeated on the same mouse over a period of 1 year and during dietary exposure to pesticide cocktails.
6) In vivo electroencephalographic exploration to determine biomarkers of aberrant neuronal activity, possibly linked to phenotypical behavioral changes.
7) Detailed histological analysis of hepatic and brain structures and cells using immunofluorescent staining and confocal microscopy
8) In vitro culture of hepatic and brain cells to study the impact of individual contaminants of the cocktail.

We here provide a succinct list of major results. Impact of pesticides:
1) Exposure to pesticide cocktail in the diet leads to distinct and topographically defined histological signs of neuro-inflammation in vivo. 2) Exposure to pesticide cocktail in the diet does not lead to histological signs of pericyte-vascular modifications in vivo. 3) In vivo and longitudinal neurophysiological measures (behavioral testing and electro-encephalography) during pesticide cocktail exposure (2 and 8 months) reveals biomarkers potentially linked to neuronal dysfunction. 4) Human brain endothelial cells and pericyte directly exposed to pesticides (mixed or single) display signs of modified growth.
Implication of the constitutive androstane receptor (CAR) in mediating (some) of the above mentioned modifications:
5) Lack of CAR protects from the obesogenic effects of pesticide dietary and long-term exposure in vivo. 6) High levels of corticosterone in CAR KO mice could underscore behavioral changes in vivo.
Thanks to HepatoBrain we are rapidly expanding our research by: i) asking for / obtaining parallel funds to add novel technical approaches to this investigation (e.g., zebrafish); ii) including and initiating key collaborations with translational-clinical programs addressing the effects of environmental contaminants exposure in humans.

HepatoBrain has created the adequate research milieu and a strong collaborative relationship between the IGF (CNRS/INSERM) and the INRA-Toxalim Units. This partnership is novel and necessary to integrate the large array of expertise and accessibility to resources that are required to study a complex problem. This partnership translates into the assessment of a public health issue by using relevant environmental molecules, at relevant dose ranges, and analyzing end-points at the systemic (liver) and brain levels. The perspective and potential development of our research efforts are significant: i) we will determine, for the first time, whether current pesticide usage protocols are safe or not; ii) we will determine possible metabolic-blood biomarkers or behavioral-electroencephalographic signatures in vivo that could be used for the diagnosis of pathological exposure to environmental contaminants; iii) from a mechanistic stand point, we will elucidate the involvement of specific hepatic nuclear receptors in mediating the effects potentially triggered by the continuous exposure to pesticides. Our research and ongoing protocols can be immediately adapted to study a broader range of environmental contaminants, including endocrine disruptors, glyphosate, etc. We are collaborating with research group dealing with contaminant monitoring in subjects and populations.

We have presented our research at specialized meetings and one manuscript is currently under consideration in Diabetes. Two manuscripts are anticipated for the end on the year.

A link between recurrent exposure to low-dose environmental toxins (pesticides) and lingering peripheral-neurovascular changes is suspected although not experimentally confirmed. This is important as it is assumed that the blood-brain barrier limits the entry of pesticides into the brain from the peripheral circulation, with the exception of episodes of acute intoxications (e.g., warfare) when sudden neurological symptoms are reported. Unrecognized mechanisms and consequences of environmental toxicity due to lingering effects of low-dose pesticide cocktails are of public health concern.
Specific nuclear receptors (NR) were proposed as mediators of environmental toxicity. A molecular entry point in our proposal is the constitutive androstane receptor (CAR or NR1I3), a key regulator of xenobiotic biotransformation. Our recently published results and preliminary data, obtained using a global CAR knock-out mouse model, unveiled coexistence of hepatic metabolic dysfunction and neurovascular changes (HepatoBrain) at adulthood stages. The phenotype includes metabolic disorder, hepatic steatosis and glucose intolerance in CAR-/- mice. We reported that peripheral pathological modifications are concomitant to increased cerebrovascular-barrier permeability, signs of neurovascular inflammation associated to lingering memory deficits and reduction of electroencephalographic theta rhythms in adult mice. These results were obtained using a global CAR KO mouse model; whether hepato-specific CAR invalidation is sufficient to drive HepatoBrain pathological changes is unknown. Known CAR modulators are pesticides. Our recent results indicated low-dose pesticide cocktails are harmful. We found that, in mice, dietary exposure to a specific pesticide cocktail at concentrations commonly found in apples (EFSA report) elicited peripheral metabolic disturbance, e.g., overweight, hepatic triglyceride accumulation, sign of inflammation and glucose intolerance. The observed changes are amongst the reported risk factors for neurovascular dysfunction. Whether, under these conditions, neurovascular pathology exists is unknown. We will evaluate the following:

WORK PACKAGE 1: HEPATO-SPECIFIC CAR INVALIDATION PROMOTES METABOLIC DISORDERS LEADING TO NEURO-VASCULAR CHANGES IN VIVO.
WORK PACKAGE 2: LOW-DOSE ENVIRONMENTAL CHEMICALS PROMOTE LONGITUDINAL HEPATOBRAIN PATHOPHYSIOLOGY.
WORK PACKAGE 3: ROLE OF CAR IN PESTICIDES COCKTAIL INDUCED HEPATOBRAIN PATHOLOGY

Invalidation or extra-physiological modulation of CAR could represent a contributing mechanism of metabolic, endocrine and neurovascular inflammatory disorders, relevant to dietary exposure to pesticide cocktails. The dynamics of hepatic-to-neurovascular long-term pathology installing upon chronic pesticide exposure have a direct social impact, inasmuch we will test a pesticide cocktail found in apples and at low dosage (EFSA report). Our study is consistent with toxicity-amplification of pesticides cocktails. We will determine whether a mechanistic link to a specific nuclear receptor (CAR) exists. We will advance towards the development of possible blood and neurophysiological biomarkers of HepatoBrain dysfunction. Unveiling a role of inflammation in the HepatoBrain axis will disclose possible therapeutic maneuver to control liver and neurovascular pathophysiology.