Glyphosate-based herbicides are the most widely used across the world; they are commercialized in different formulations. Their residues are frequent pollutants in the environment. In addition, these herbicides are spread on most eaten transgenic plants, modified to tolerate high levels of these compounds in their cells. Up to 400ppm of their residues are accepted in some feed.  

We exposed human liver HepG2 cells, a well known model to study xenobiotic toxicity, to four different formulations and to glyphosate, which is usually tested alone in chronic in vivo regulatory studies. We measured cytotoxicity with three assays (Alamar Blue((R)), MTT, ToxiLight((R))), plus genotoxicity (comet assay), anti-estrogenic (on ERalpha, ERbeta) and anti-androgenic effects (on AR) using gene reporter tests. We also checked androgen to estrogen conversion by aromatase activity and mRNA.  

All parameters were disrupted at sub-agricultural doses with all formulations within 24h. These effects were more dependent on the formulation than on the glyphosate concentration. First, we observed a human cell endocrine disruption from 0.5ppm on the androgen receptor in MDA-MB453-kb2 cells for the most active formulation (R400), then from 2ppm the transcriptional activities on both estrogen receptors were also inhibited on HepG2. Aromatase transcription and activity were disrupted from 10ppm. Cytotoxic effects started at 10ppm with Alamar Blue assay (the most sensitive), and DNA damages at 5ppm.  

A real cell impact of glyphosate-based herbicides residues in food, feed or in the environment has thus to be considered, and their classifications as carcinogens/mutagens/reprotoxics is discussed. 

Reference: Gasnier C, Dumont C, Benachour N, Clair E, Chagnon MC, Séralini GE., Glyphosate-based herbicides are toxic and endocrine disruptors in human cell lines, University of Caen, Institute of Biology, Lab. Biochemistry, Caen, France, Toxicology. 2009 Jun 16.

Although it is known that infants are more susceptible than adults to the toxic effects of pesticides, this increased vulnerability may extend much longer into childhood than expected, according to a new study by researchers at the University of California, Berkeley.

Among newborns, levels of paraoxonase 1 (PON1), an enzyme critical to the detoxification of organophosphate pesticides, average one-third or less than those of the babies’ mothers. It was thought that PON1 enzyme activity in children approached adult levels by age 2, but instead, the UC Berkeley researchers found that the enzyme level remained low in some individuals through age 7.

Based upon the findings, reported this month in the journal Environmental Health Perspectives, the study authors recommend that the U.S. Environmental Protection Agency (EPA) re-evaluate the current standards for acceptable levels of pesticide exposure.

“Current EPA standards of exposure for some pesticides assume children are 3 to 5 times more susceptible than adults, and for other pesticides the standards assume no difference,” said Nina Holland, UC Berkeley adjunct professor of environmental health sciences and senior author of the paper. “Our study is the first to show quantitatively that young children may be more susceptible to certain organophosphate pesticides up to age 7. Our results suggest that the EPA standards need to be re-examined to determine if they are adequately protecting the most vulnerable members of the population.”

In 2001, the EPA began restricting organophosphate pesticides in products sold for use in homes, mainly because of risks to children. However, organophosphate pesticides, such as chlorpyrifos and diazinon, are still used in agriculture in the United States and elsewhere.

The study, conducted by UC Berkeley’s Center for the Health Assessment of Mothers and Children of Salinas (CHAMACOS), involves 458 children from an agricultural region who were followed from birth through age 7. Cord blood samples were collected from all children to determine their PON1 genotype and to obtain baseline measures of the enzyme’s activity level.

For more than 100 of the children in the study, researchers were able to obtain at least four additional measurements – at ages 1, 2, 5 and 7 – of PON1 activity. Almost all the children in the study had 2 to 3 time points assessed, for a total of 1,143 measurements of three types of PON1 enzyme activity.

One’s PON1 genotypic profile determines how effectively the enzyme can metabolize toxins. For example, people with two copies of the Q form of the gene – known as a QQ genotype – produce a PON1 enzyme that is less efficient at detoxifying chlorpyrifos oxon, a metabolite of chlorpyrifos, than the enzyme produced by people with two R forms of the gene. Similarly, individuals with two T forms of the PON1 gene on a different part of the chromosome generally have a lower quantity of the enzyme than do those with two C forms of the gene.

Previous research led by Holland found that some of the QQ newborns may be 50 times more susceptible to chlorpyrifos and chlorpyrifos oxon than RR newborns with high PON1 levels, and 130 to 164 times more susceptible than some of the RR adults.

Of the children in this latest study, 24 percent had the QQ genotype, and 18 percent had the TT genotype, both of which are associated with lower activity of the PON1 enzyme. Moreover, 7.5 percent of the children had both QQ and TT genotypes, which is considered an even more vulnerable profile.

On average, the quantity of enzyme quadrupled between birth and age 7. The greatest rise in enzyme activity was among children with the RR and CC variants of the PON1 gene, which quickly outpaced the increase in children with the QQ and TT genotypes.

The fact that enzyme activity remained low for certain kids with vulnerable genotypes well past age 2 was surprising for the study authors. The researchers are continuing to collect data for these children as they grow older to see if the pesticide susceptibility continues.

“In addition to its involvement in the metabolism of pesticides, many studies are now finding that PON1 may play an important role in protecting against oxidative stress, which is linked to diseases from asthma to obesity and cardiovascular disease,” said study lead author Karen Huen, a UC Berkeley Ph.D. student in environmental health sciences. “The children in our study whose genotypes are related to lower PON1 activity may not only be more susceptible to pesticides throughout much of their childhood, they may also be more vulnerable to other common diseases related to oxidative stress.”

Notably, other studies have found that PON1 genotypes vary by race and ethnicity, with the Q variants more common among Caucasians, less common among Latinos, and least common among African Americans. The majority of the subjects in this study were Mexican-American.

“What’s important about this study is that it shows that young children are potentially susceptible to certain organophosphates for a longer period of time than previously thought,” said Brenda Eskenazi, UC Berkeley professor of epidemiology and director of CHAMACOS and the Center for Children’s Environmental Health Research. “Policymakers need to consider these vulnerable populations when establishing acceptable levels of exposure to different pesticides.”

Funding from the National Institute of Environmental Health Sciences and the EPA helped support this research.

Reference: UC Berkeley, Children susceptible to pesticides longer than expected, study finds, June 22, 2009

Based on New Harvard Study, Tobacco Control Researchers Call on FDA to Require Complete Disclosure from Tobacco Companies of Changes Made to Cigarettes 

Boston, MA — As President Obama prepares to sign a bill giving the Food and Drug Administration (FDA) oversight of the tobacco industry, a new study from Harvard School of Public Health (HSPH) researchers shows that tobacco manufacturers have continually changed the ingredients and the design of their cigarettes over time, even if those changes have exceeded acceptable product variance guidelines. The result, say the researchers, is that consumers who buy the same brand of product are not made aware of how that product has been altered and what effect those alterations might have on their levels of addiction or harm.  

“I hope the FDA requires disclosure of any changes made to tobacco products and that the changes are disallowed if shown to increase appeal, addiction and harm,” said Greg Connolly, director of the Tobacco Control Research Program at HSPH.

The study appears in the “Online First” section of the Journal of Tobacco Control and will appear in an upcoming print issue of the journal.  

For their study, Connolly and lead author Geoffrey Ferris Wayne, an HSPH researcher, studied internal tobacco company documents released following the 1998 Master Settlement Agreement. These documents describe significant changes made to commercial products over time, including blend, processing, casing, flavoring and physical design features. For example, new methods were developed to process tobacco, altering the smoke chemistry and the form of nicotine delivery, and the levels of processed tobaccos were regularly adjusted within brands.  

Despite the constant innovation of tobacco products, which in many cases have exceeded the levels of acceptable variance established within the tobacco industry, for the most part, these changes were not disclosed to consumers, say the researchers. 

“Even incremental changes that occur over a period of years can result in significant design differences. The resulting product may have altered chemistry or delivery, yet the smoker is largely unaware of these changes. This underscores the need for industry transparency and accountability,” said Ferris Wayne.

The study builds on earlier research done at HSPH on how products are designed to enhance appeal and addiction. At Senate hearings on the FDA bill last year, Connolly discussed that research, including how tobacco companies have increased nicotine content over time, manipulated menthol and added candy-like flavors to enhance appeal to children.  

Until regulators have a system in place for assessing product revisions, Connolly and Ferris Wayne advise that all changes to tobacco products be reported to the FDA and that no changes be allowed until they have been scientifically shown to reduce addiction or harm.  

The study was supported by the National Cancer Institute.  

References:  Harvard, Based on New Harvard Study, Tobacco Control Researchers Call on FDA to Require Complete Disclosure from Tobacco Companies of Changes Made to Cigarettes, , Friday, June 19, 2009 

“Regulatory Assessment of Brand Changes in the Commercial Tobacco Product Market,” Geoffrey Ferris Wayne, Greg Connolly, Journal of Tobacco Control, online June 14, 2009.

Tobacco is an important cash crop of Pakistan. Pesticides are commonly used to increase the crop yield, but their health impact has not been studied yet.  

The objectives of the study were to determine the frequency of pesticide poisoning and to explore the knowledge, attitudes and practices (KAP) towards safety measures among the tobacco farmers in Swabi, Pakistan.  

One hundred and five tobacco farmers involved in pesticide application were randomly selected from two villages of district Swabi. A structured questionnaire was used for clinical and KAP information. Plasma cholinesterase (PChE) levels were measured by Ellman’s method by using GD Italy kits. All tobacco farmers were males with a mean (SD) age of 26 (9) years.  

The majority of the farmers reported multiple symptoms headache, dizziness, vomiting, shortness of breath, muscle weakness and skin rash correlate with the clinically significant depression of PChE levels. 

Out of 105 pesticide applicators, 58 (55%) had post-exposure reduction in PChE levels <20% from baseline, 35 (33%) had mild poisoning (20-40% reduction) and 12 (11%) had moderate poisoning (>40% reduction).  

Most of the farmers did not use any personal protective equipment during pesticide handling. Only a few used shoes (31%), masks (14%) and gloves (9%) during pesticide spray.  

In conclusion, the tobacco farmers had mild to moderate pesticide poisoning, which was correlated with depression in PChE levels. Moreover, most farmers had little knowledge about the safety measures, casual attitude and unsatisfactory safety practices with regard to the use of basic protective equipments during pesticide applications on the tobacco crop. 

Reference: Khan DA, Shabbir S, Majid M, Naqvi TA, Khan FA., Risk assessment of pesticide exposure on health of Pakistani tobacco farmers, aDepartment of Pathology, Army Medical College, NUST, Rawalpindi, Pakistan, Journal of Exposure Science and Environmental Epidemiology advance online publication, 17 June 2009; doi:10.1038/jes.2009.13.

The authors investigated the association of early-life exposure to indoor air pollution with neuropsychological development in preschoolers and assessed whether this association differs by glutathione-S-transferase gene (GSTP1) polymorphisms. A prospective, population-based birth cohort was set up in Menorca, Spain, in 1997-1999 (n = 482).

Children were assessed for cognitive functioning (McCarthy Scales of Children’s Abilities) and attention-hyperactivity behaviors (Diagnostic and Statistical Manual of Mental Disorders, 4th Edition) at age 4 years.

During the first 3 months of life, information about gas appliances at home and indoor nitrogen dioxide concentration was collected at each participant’s home (n = 398, 83%). Genotyping was conducted for the GSTP1 coding variant Ile105Val. Use of gas appliances was inversely associated with cognitive outcomes (beta coefficient for general cognition = -5.10, 95% confidence interval (CI): -9.92, -0.28; odds ratio for inattention symptoms = 3.59, 95% CI: 1.14, 11.33), independent of social class and other confounders.

Nitrogen dioxide concentrations were associated with cognitive function (a decrease of 0.27 point per 1 ppb, 95% CI: -0.48, -0.07) and inattention symptoms (odds ratio = 1.06, 95% CI: 1.01, 1.12).

The deleterious effect of indoor pollution from gas appliances on neuropsychological outcomes was stronger in children with the GSTP1 Val-105 allele. Early-life exposure to air pollution from indoor gas appliances may be negatively associated with neuropsychological development through the first 4 years of life, particularly among genetically susceptible children.

Reference: Morales E, Julvez J, Torrent M, de Cid R, Guxens M, Bustamante M, Künzli N, Sunyer J., Association of early-life exposure to household gas appliances and indoor nitrogen dioxide with cognition and attention behavior in preschoolers, Center for Research in Environmental Epidemiology, Barcelona, Catalonia, Spain, Am J Epidemiol. 2009 Jun 1;169(11):1327-36.