Detrimental effects of 6 months exposure to very low doses of a mixture of six pesticides associated with chronic vitamin deficiency on rats

https://doi.org/10.1016/j.fct.2021.112188Get rights and content

Highlights

  • Vitamin deficiency modified the effects of a low-dose pesticides' mixture in rats.

  • The combined stressors resulted in abnormal serum lipid profile and increased ALKP.

  • The combined stressors raised activity of CYP1A1, CYP1A2, CYP2B1 and GST in liver.

  • The combined stressors decreased erythrocytes count, hematocrit and hemoglobin levels.

  • Neither the pesticide mixture nor vitamin deficiency alone induced relevant changes.

Abstract

This study aimed to evaluate the long-term low-dose effects of exposure to a mixture of 6 pesticide active substances (diquat, imazamox, imazethapyr, tepraloxydin, bentazone, acifluorfen) and to elucidate if chronic vitamin deficiency can influence their toxicity. Two hundred Wistar rats were divided in 4 groups: a vitamin-sufficiency control group, a vitamin-deficiency control group, a vitamin sufficiency test group and a vitamin-deficiency test group. The test groups were treated with the aforementioned pesticides at doses 100 times lower than the corresponding NOAEL. After 6 months, ten rats from each group were sacrificed and a complete evaluation of blood and urine biochemistry, biomarkers of oxidative stress, xenobiotic detoxification enzymes and lysosomal enzymes and organ histopathology was performed. The pesticides mixture and vitamin deficiency determined an increase in alkaline phosphatase levels and urinary calcium levels, abnormal serum lipid profile, and a decrease of total blood proteins levels, red blood cells, haematocrit and haemoglobin. The combination of the two stressors up-regulated CYP1A1, CYP1A2, CYP2B1 and GST levels. This study provides a new proof for the need to move forward from single chemical testing to a more complex approach to account for the multitude of stressors that can challenge the setting of real safety levels.

Introduction

In the last years, the industrialization and modernization of the production processes in all sectors entails omnipresent exposure to a cocktail of chemicals. Everything that we eat, drink, breath or use as hygiene and lifestyle products contains mixtures of chemicals. From early intrauterine life till elderly, the individual is continuously exposed to chemicals with beneficial or detrimental effects depending on the doses, windows of exposure and combinations. Many of these exposures are considered risk factors for many diseases, and where associated with genetic patterns can contribute to the occurrence of diseases (Docea et al., 2017). The organism can respond differently to these stressors with time-dependent variations that can vary from changes intended to maintain the homeostasis of the organism to pathological changes associated with different shapes of dose-response curves (Agathokleous, 2018; Agathokleous and Calabrese, 2020). Till now the toxicological evaluation of chemicals has been focused mainly on testing high doses of single chemicals in experimental animals for setting safety limits and to identify target organ(s) (Tsatsakis et al., 2016). Unfortunately, this approach does not mirror the real-life exposure scenario in which individuals are exposed simultaneously to low doses of multiple stressors (Tsatsakis et al., 2016). The safety evaluation of mixtures has been done till now only for commercial mixtures and for mixtures of chemicals that share the same mode of action, the same phenomenological effect or target organ, but they don't incorporate the dose-response spectrum and the temporal variations that can better predict the time-dependent dose responses to environmental relevant mixtures. Studies have shown that mixtures of xenobiotics can lead not only to predictable additive effects but also to unpredictable synergistic, or antagonistic effects (Lukowicz et al., 2018; Mesnage et al., 2020) (Hernández et al., 2013; Docea et al., 2018, 2019; Tsatsakis et al., 2019a, 2019b, 2019c; Fountoucidou et al., 2019; Sergievich et al., 2020). These effects cannot be predicted by standard single chemical toxicological evaluations and need a more complex approach for protecting the population. Starting from these limitations inherent to single-chemical testing, new methodologies have been proposed for simulating the real-life exposure scenario in which individuals are exposed to multiple stressors from different sources on a long term regimen (Docea et al., 2016; Goumenou and Tsatsakis, 2019; Hernández and Tsatsakis, 2017; Tsatsakis et al., 2017, 2019d; Tsatsakis and Lash, 2017).

Environmental or occupational exposure to plant protection products has been associated with a full range of chronic disorders, such as hepatic diseases (Freire et al., 2015), haematological diseases (Freire et al., 2015; García-García et al., 2016), renal diseases (Georgiadis et al., 2018a; Năstăsescu et al., 2020), cardiovascular diseases (Georgiadis et al., 2018b), obesity and metabolic syndrome (Petrakis et al., 2017), neurological diseases (Aloizou et al., 2020), infertility (Sifakis et al., 2017) and even cancers (Vakonaki et al., 2013; Dolapsakis et al., 2001). These findings can be explained by the fact that the so-called safety levels are set based on single compound experiments that follow one critical effect, while in reality humans are exposed to a cocktail of chemicals that, depending on the dose, can modify the effects or lead to different target organs effects (Hernández et al., 2013).

B vitamins and folate are implicated in a lot of functions of the organism and their deficiency has been associated with an increased risk for cardiovascular disorders, degenerative diseases, immune dysfunctions and inflammatory diseases (Mikkelsen and Apostolopoulos, 2018). Vitamin B2 or riboflavin deficiency can alter iron absorption and has been implicated in metabolic disorders associated with impaired tryptophan metabolism and mitochondrial function (Thakur et al., 2017). Vitamin B3 or niacin is an indispensable vitamin that plays a role in more than 400 NAD(P) (nicotinamide adenine dinucleotide phosphate)-dependent reactions in all areas of human metabolism. The deficiency of this vitamin is associated with genome instability, apoptosis, chromosomal breakage, telomere erosion and cancer development (Kirkland, 2012). Vitamin K or menadione is a cofactor of γ-glutamyl carboxylase involved in the activation of a family of vitamin K dependent-proteins. These proteins have several functions in the organism related to reproduction, bone and vascular function and metabolism (Fusaro et al., 2020). Studies have shown that vitamin K deficiency is followed by a decrease of matrix Gla protein (a vitamin K-dependent protein that inhibits vascular calcification), thus increasing the risk of cardiovascular diseases (Piscaer et al., 2017).

Starting from the new methodology for the toxicological testing of chemical mixtures under the real-life risk simulation approach proposed by Tsatsakis et al. (2017), this study aimed to evaluate the long-term effects of exposure to a mixture of 6 frequently used pesticides (diquat, imazamox, imazethapyr, tepraloxydin, bentazone, and acifluorfen) in low doses (100 times below their corresponding critical NOAELs). These chemicals were selected as they are herbicides usually found as residues in food and drinking water (A)A: Overview of Sod (2002); Authority (2017); Agency (2011). Diquat mechanism of toxicity in humans is associated with generation of oxidative stresss eventually leading to hepatotoxicity, neurotoxicity and nephrotoxicity (Magalhães et al., 2018a). Imazamox is considered safe to non-target species, showing low toxicity in animal studies with the critical effect being decrease in bodyweight gain and the decrease in food consumption (Authority, 2016). The long-term effects of imazethapyr are not completely known, even if the general population can be exposed through food or water route (Koutros et al., 2009; Imazethapyr; Pesticide Tolerance, 2002). The main target organs for tepraloxydim toxicity were the liver, the spleen/hematopoietic system and reproductive system (Federal Register/Vol. 7, 2011) Bentazone has been associated with impairment of blood coagulation and liver and kidney effects in regulatory studies (Authority, 2015a) Acifluorfen is associated with liver and kidney toxicity as well as teratogenicity and carcinogenicity in non-target organisms (Kenyon and Duke, 1985) and the proposed mechanisms underlying cytotoxicity in humans include activation of nuclear receptors (CAR and PPARα) whereas inhibition of protoporphyrinogen oxidase inhibition each may play some role in rodent liver changes (Kuwata et al., 2016) In order to simulate more the real-life exposure scenario, plant protection products were used instead of pesticide active substances. This study also addressed whether chronic vitamin deficiency can influence the toxicity of the pesticides mixture.

Section snippets

Animal experiment

This article presents part of the results of a larger study whose methodology was previously described by Tsatsakis et al. (2019b). Briefly, 200 male Wistar rats, 30 days old, from Affiliated Unit “Stolbovaya” of Scientific Center for Biomedical Technology of the Federal Medical and Biological Agency, Moscow, Russia, were divided into 4 experimental groups (50 rats each):

  • -

    C-100 group – or the control group that received the standard rat diet (AIN-93) that contained 100% of the dose of

Weight gain of rats over time

All the growth models showed a very good fit (adjusted R-squared of ≥0.992). The pesticide mixture (T100 group vs C100 group) caused a statistically significant decrease in Wmax (maximum weight) and inflection time (p < 0.001 for both), and a statistically significant increase in the growth rate (Table 2). Decreased vitamin consumption (C25 group vs C100 group) caused a statistically significant decrease in Wmax (p < 0.001) and in the growth rate (p = 0.020) but it had no effect on the

Discussion

This study evaluated the effects of six months exposure of rats to a mixture of six pesticides at doses corresponding to their individual NOAEL/100 (for the selected pesticides it corresponded to their ADI) for many that are considered safe for consumers. In the same time, we sought whether vitamin deficiency may modify the detrimental effects of the pesticide mixture on a rat model. The wide spectrum of biological functions modulated by B vitamins, vitamin K and folates determines that

Conclusions

Six-month exposure of rats to a mixture of diquat, imazamox, imazethapyr, tepraloxydin, bentazone and acifluorfen, at doses 100 times below their corresponding NOAELs, together with a vitamin-deficient diet (B vitamins, folates and vitamin K) resulted in increased ALKP levels, abnormal serum lipid profile (decreased triglycerides and cholesterol and increased lipase), decreased total blood proteins levels, and increased urinary calcium levels. The combination the two stressors also decreased

Funding

The research was conducted with the financial support of the Ministry of Science and Higher Education of the Russian Federation (research project No. 0529-2019-0056).

CRediT authorship contribution statement

Aristidis Tsatsakis: Conceptualization, Methodology, Investigation, Writing – review & editing, Supervision. Nadezhda V. Tyshko: Methodology, Investigation, Writing – review & editing, Supervision. Marina Goumenou: Conceptualization, Methodology, Investigation, Writing – review & editing, Supervision. Svetlana I. Shestakova: Investigation, Data curation, Writing – original draft. El'vira O. Sadykova: Investigation, Data curation, Writing – original draft. Valentin M. Zhminchenko: Investigation,

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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