ISIS
Steep decline in human male sperm count concomitant with rise in testicular germ cell cancer, congenital malformations of the male reproductive tract and drop in serum testosterone levels, all pointing towards increasing exposure to glyphosate/Roundup herbicides during the past decades, now corroborated by lab findings Dr Mae-Wan Ho
A
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“The infertility timebomb: are men facing extinction?”
The headline of a newspaper article published in 2010 [1] refers to findings from decades of research carried out by Niels Shakkebaek, a professor at University of Copenhagen. Male infertility has been rising sharply in industrialized countries worldwide, one in five healthy men between the ages of 18 and 25 produce abnormal sperm counts. The problems start in the womb, says Dr. Gillian Lockwood, medical director of Midland Fertility Services in the UK. Testis development begins in the growing foetus. Factors blamed include too much beef in the diet rich in polycyclic aromatics, obesity during pregnancy, exposure to smoke, pesticides, traffic fumes, plastics and even soybeans.
Shakkebaek first highlighted the issue during a mini symposium at the European Medical Research Councils plenary meeting in Strasbourg in 2009. Semen quality has been declining in the past half century. In men without fertility problems, average sperm count dropped from 113 x 106 to 66 x 106/ml. About 20 % of young men in various European countries have sperm counts below the WHO (World Health Organization) reference level of 2o m/ml, and 40 % of have levels below 40 m/ml associated with prolonging the time to pregnancy [2]. Concomitantly, the demand for assisted reproductive technology (ART) is growing. In Denmark, more than 7 % of all children born in 2007 were conceived using ART.
There are geographical differences in semen quality. Finnish men have 35 % higher sperm counts than Danish men, while Scottish and French sperm counts are in between. Japanese sperm counts are as low as those of the Danes, and Singapore men have even lower sperm counts.
The trend in semen quality has implications for health in general, as men with poor semen quality seem to have increased mortality rates and shorter life expectancy. Infertility is also closely linked to several dysfunctions and abnormalities of male reproductive organs that have been rising concomitantly with infertility.
Infertility trend associated with testicular germ cell cancer, congenital malformations & low testosterone
Testicular germ cell cancer (TGC) is the commonest cancer in young men in many countries, associated with impaired semen quality and lower fertility rates even prior to cancer development. The incidence of TGC has been increasing over the past 40 to 50 years in the majority of industrialized countries coincidentally with the declining trend in semen quality. TGC is initiated during foetal development. The regional differences in TGC incidence in Europe follow the same pattern as observed for semen quality.
Congenital malformations of the male reproductive tract – undescended testis and incomplete fusion of the urethral folds that form the penis – are among the most frequent congenital malformations in human males. These two abnormalities share common risk factors, both associated with reduced fertility; the first malformation is also associated with poor semen quality and considerably increased risk of TGC. Incidences of these malformations appear to have been increasing in the Western world over recent decades.
Testosterone, the male hormone, is the major driver of male reproductive development and function. Suppression of its levels within the adult testis shuts down spermatogenesis and induces infertility. Studies of men with idiopathic infertility – for which the cause is unknown - and low sperm counts often show evidence of abnormal Leydig cells, which produce testosterone in the testis.
In Europe, incidences of TGC and congenital reproductive tract malformations have been going up coincidentally with a downward trend in semen quality and testosterone levels (although there are only data for the latter in Denmark). These disorders share common risk factors and are risk factors for one another. Consequently, it has been proposed that the conditions collectively may represent a syndrome - a testicular dysgenesis syndrome (TDS) - caused by a common underlying causal factor, which is either a change in lifestyle or an environmental toxin, especially endocrine disrupting chemicals such as pesticides. Notably, the review published by the European Science Foundation (an official body that coordinates international research programmes in Europe) fails to mention glyphosate explicitly, even though its use has been rising most rapidly among pesticides in Europe and in the rest of the world since the 1980s to 1990s.
Age-independent testosterone decline reflects rise in glyphosate use with GM crops
In America, there has been a substantial age-independent decline in testosterone that does not appear attributable to observed changes in explanatory factors including health status and lifestyle characteristics such as smoking and obesity. The estimated declines were larger than the cross sectional declines typically associated with age, as shown in Figure 1 [3].
The data are from randomly selected men living in greater Boston, Massachusetts in the United States, not connected with studies on infertility but with aging in general, as considerable loss of serum testosterone is thought to be a mark of male aging.
It is notable that the steep decline in testosterone levels began just after the introduction of genetically modified (GM) crops in 1994 with concomitant increase in glyphosate herbicides use on glyphosate tolerant GM crops. A comprehensive review article has blamed glyphosate for “most of the diseases and conditions associated with a Western diet” including infertility [4], although the precise mode of action, at least in the case of infertility, remains unclear.
Roundup more damaging than glyphosate
There is already evidence that glyphosate is an endocrine disrupting chemical (see later), but the extent of the problem is far greater than it appears.
Different glyphosate formulations vary in toxicity, mainly because some of them contain adjuvants that are either toxic by themselves, or else exert synergistic effects with glyphosate. It has long been known that Monsanto’s formulation Roundup, the most widely used glyphosate herbicide, is far more damaging than glyphosate itself (reviewed in [5]
Ban GMOs Now, ISIS special report).
Giles-Eric Séralini and colleagues at University of Caen in France clearly demonstrated that POEA (polyethoxylated tallowamine, a major adjuvant surfactant in Roundup) alone was by far the most cytotoxic for several human cell types, at concentrations a hundredth to ten-thousandth that of glyphosate itself and other formulations without POEA [6]. Another study from the same laboratory also showed that Roundup exposure damages testosterone producing Leydig cells from mature rat testis at concentrations a tenth of agricultural use and beginning 1 hour after exposure [7]. Within 24-48 h, the same formulation was toxic to other cells inducing cell death, in contrast to glyphosate alone, which is only toxic to Sertoli cells (feeder cells for germ cells). At 48 h, Roundup induces apoptosis (programmed cell death involving DNA fragmentation) in germ cells and in Sertoli/germ cells co-culture. At the very low, non-toxic concentration of 1 ppm, both Roundup and glyphosate decreased testosterone level by 35 %. These experiments expose a major inadequacy in the regulatory regime, which still regards POEA in Roundup as an inert adjuvant for which no risk assessment is required.
A recent laboratory experiment shows that Roundup has direct, acute impacts on the mammalian testis at levels of exposure orders of magnitude below recommended agricultural concentrations.
Acute Roundup exposure at very low concentrations kills cells in the immature testis
The Brazilian research team led by Ariane Zamoner at the Federal University of Santa Catarina in Florianópolis, and Federal University of Rio Grande do Sul in Porto Alegre, are well aware of the increased toxicity of Roundup compared with glyphosate, and were prompted to investigate the effects of Roundup by the high prevalence of reproductive dysfunction among agricultural workers occupationally exposed to the herbicide. They looked at concentrations of Roundup 2 to 3 orders of magnitude below the 10 000 to 20 000 ppm (10-20g/L) used in agriculture, which is quite realistic in terms of exposure levels for agricultural workers and members of the general public close to or within the spraying range [8].
The researchers found that brief exposure to Roundup at 36 ppm (0.036 g/L) for 30 minutes was sufficient to induce oxidative stress (a failure of energy metabolism, see later) and activate multiple stress-response pathways leading to cell death in the pre-puberty rat testis.
The team concluded [8]: “Altogether, the Ca2+-mediated disturbances by glyphosate-Roundup in rat testis cells around 36 ppm, could contribute to the reproductive effects observed in male agricultural workers exposed to this pesticide at prepubertal age.”
Detailed mechanisms of action identified
The team found that Roundup increases intracellular Ca2+ concentration by opening L-type -voltage-dependent Ca2+ channels – thereby allowing Ca2+ to enter the cells - as well as targeting the endoplasmic reticulum IP3 (inositol triphosphate) and ryanodine receptors (both Ca2+ release channels), leading to Ca2+ release and overload within the cells, setting off cell death. The mechanisms involved were inferred from experiments with specific inhibitors that cancelled out the effect of Roundup as well as Ca2+ influx; and confirmed by the increase in radioactive tracer 45Ca2+ uptake by testis incubated with Roundup at 36 ppm. These events were prevented by the antioxidants Trolox and ascorbic acid, which counteract the reactive oxygen species (see below) responsible for the oxidative stress. Activated protein kinase C, phosphatidylinositol 3-kinase, and the mitogen-activated protein kinases such as ERK1/2 and p38MAPK all play a role in eliciting Ca2+ influx and cell death.
Roundup also decreases the levels of reduced glutathione (GSH, the tissue’s own antioxidant) as consistent with oxidative stress, and increases the amounts of thiobarbituric acid reactive species (TBARS) and protein carbonyls, which are signs of oxidative damage from reactive oxygen species to lipids and proteins respectively. Exposure to Roundup stimulates the activities of a whole collection of enzymes supporting the down-regulation of GSH levels.
The research team looked at acute Roundup exposure of both whole immature Wistar rat testis and isolated Sertoli cells in culture; and the findings were very similar in the two systems.
Based on their experimental results, the team propose that Roundup toxicity is due to Ca2+ overload, resulting in cell signalling fault, a stress response and/or defence against depleted antioxidant, all contributing to the death of Sertoli cells, thereby impacting on male fertility.
The new findings are consistent with the well-known involvement of Ca2+ in cell death from oxidative stress. Oxidative stress causes Ca2+ influx into the cytoplasm from the extracellular environment and from the endoplasmic reticulum [9]. Rising Ca2+ concentration in the cytoplasm in turn causes Ca2+influx into the mitochondria and nuclei. In the mitochondria, Ca2+ accelerates the disruption of normal oxidative metabolism leading to necrotic cell death. In nuclei, Ca2+ modulates gene transcription and nucleases that control apoptosis (programmed cell death that involves fragmentation of DNA).
There is already evidence that glyphosate may act as an endocrine disruptor for both males and females by altering aromatase activity, oestrogen regulated genes, and testosterone levels in rats [10]. But Roundup acts via different mechanisms. Roundup exposure during pregnancy and lactation at a level that did not induce maternal toxicity in Wistar rats nevertheless induced adverse reproductive effects in male offspring, including decreased daily sperm production during adulthood, increase in abnormal sperms, and low testosterone serum level at puberty. In exposed female offspring, only a delay in vaginal canal opening was observed [11].
Oxidative stress and endocrine disrupting effects specific to Roundup
The key to understanding the action of Roundup on male infertility is the reactive oxygen species (ROS) generated in oxidative stress (see [12, 13]
The Body Does Burn Water and
Living with Oxygen, SiS 43). Not only are ROS implicated in practically every chronic human disease including cancer [14] (
Cancer a Redox Disease, SiS 54), but also play an essential role in the pathogenesis of many reproductive processes as detailed in a review published in 2003 [15]. In male-factor infertility, oxidative stress attacks the lipids of the sperm plasma membrane and the integrity of DNA in the sperm nucleus. In addition, ROS induce DNA damage, accelerate germ cell death and decrease sperm counts, thereby contributing to male infertility.
ROS is so closely linked to male infertility that infertile males generating high levels of ROS are 7 times less likely to initiate a pregnancy compared with those with low levels of ROS. A meta-analysis demonstrated that ROS levels were significantly correlated with the fertilization rate in couples undergoing in vitro fertilization [16].
Ashok Agarwal at the Center for Advanced Research in Human Reproduction, Infertility and Sexual Function, Cleveland Ohio in the United States led a retrospective study on 132 male factor infertility (MFI) patients (failure to initiate pregnancy with fertile partner after one year of unprotected sex) consisting of 24 with all normal sperm parameters, 38 with all abnormal parameter and the rest with 1 or more abnormal parameters [17]. They found that the 34 normal healthy donors (controls) had significantly higher sperm concentrations, motility and morphology compared with all MFI patients. There was a significant association between MFI and ROS with odds ratio of 4.25, independently of sperm parameters and age. They concluded that high ROS is an independent marker of MFI, irrespective of whether these patients have normal or abnormal semen parameters. They proposed that ROS measurement should be included a part of idiopathic infertility evaluation, and treatment with antioxidants may be beneficial for such patents.
ROS are generated as intermediates in the central metabolic process whereby oxygen-breathing organisms obtain energy to fuel all their activities. The energy metabolism takes place in the mitochondria, the tiny membranous powerhouses within cells where fragments from the breakdown of glucose are oxidized ultimately into carbon dioxide and water. It involves a tightly coupled process of oxidative phosphorylation in which electrons and protons are extracted from the chemical fragments, with electrons transported down the electron transport chain and protons transported up the proton gradient, so that their energy can be tapped to make ATP (adenosine triphosphate, the universal energy intermediate of the body) (for a good summary of the entire process see Chapters 21 and 22 of [18]
Living_Rainbow_H2O, ISIS publication). During this tightly coupled process, ROS are generated as partially oxidized intermediates [13]. Consequently, disturbances that uncouple oxidative phosphorylation lead to a failure of oxidation and release the partially oxidized and damaging ROS intermediates into the cell, resulting in oxidative stress.
It is very likely that the primary target of Roundup, especially its POEA surfactant, is the mitochondria, which play a key role in the development of sperm cells and sperm motility [19]. In addition, male infertility could arise from ROS damages to mitochondrial DNA.
Francisco Peixoto at University of Trás-os-Montes, Real, in Portugal compared the effects of Roundup with glyphosate on isolated rat liver mitochondria [20] and found dramatic differences. Roundup collapses the transmembrane potential of the mitochondria and uncouples oxidative phosphorylation, depressing the rates of oxidation, with effects starting at 0.5 mM (7.5 ppm). These effects are most likely due to non-specific permeation of the mitochondrial membrane by Roundup or its adjuvant POEA. In addition, Roundup specifically inhibited succinate dehydrogenase, succinate cytochrome c reductase, and ATP synthase and ATPase, key enzymes in oxidative phosphorylation. Glyphosate, on the other hand, does not have any significant effects on the function of mitochondria up to the highest concentration used, 15 mM (253.5 ppm).
