A Comparison of the Effects of Three Gm Corn Varieties on Mammalian Health Essay Example

and MON 863, which were fed to rats for 90 days. We focused on sex-related, temporal, linear, and non-linear dose effects that are often associated with the development of chronic and endocrine diseases.

The European governments have acquired and publicly released raw data for examination and evaluation. These data are utilized as a model to study potential subchronic toxicological effects of genetically modified (GM) cereals in mammals and humans. These investigations represent the longest in vivo tests carried out on mammals consuming such GMOs. The animals were observed for various blood and organ parameters. One variety of corn, known as NK 603, has been genetically modified to withstand the herbicide Roundup, meaning it contains residues of this formulation. The other two types of GM maize produce two distinct insecticides, which are modified versions of Cry1Ab (MON 810) and Cry3Bb1 (MON 863) proteins derived from Bacillus thuringiensis. Consequently, all three GM maize varieties contain new pesticide residues that will be present in food and feed products. As a result, animal feeding studies can be used to evaluate potential physiological impacts due to either the recognized mutagenic effects of the GM transformation process or the presence of the aforementioned novel pesticides within these plants.

The main objective of this study is to verify three new side effects related to the consumption of genetically modified (GM) maize, taking into account sex and dose as variables. Additionally, it aims to investigate the impact of these GM foods on the kidney and liver, which are essential organs for detoxification. It also intends to explore the correlation between sex, dose, and the development of chronic and endocrine diseases.

This research holds significance due to

the ongoing global controversy surrounding the safety and regulatory approval process of GM crops and foods. Prior to their commercialization, industries conducted confidential tests on their GM crops, pesticides, drugs or chemicals. In this study, available data was analyzed in order to compare health effects associated with consuming different GMOs. Statistical analysis was performed using measurements from 10 rats per group for blood and urine parameters. The researchers affirm that they followed OECD guidelines and standards throughout their investigation.

Only two feeding doses were tested per sex for each type of GM maize. These doses included either 11% or 33% GM maize in an otherwise equivalent equilibrated diet. In the case of the diet containing only 11% GM maize, the missing 22% was replaced with non-GM maize (specific varieties not specified). Additionally, there were two comparative control groups that were fed diets with similar amounts of the closest isogenic or parental maize variety. Furthermore, groups of animals were given diets containing one of six other normal (non-GM) reference maize lines. Although these lines were the same for the NK 603 and MON 810 tests, they differed in the MON 863 trials. It is important to note that these unrelated, different non-GM maize types were not proven to be substantially equivalent to the GMOs. It should be noted that the quantity of certain sugars, ions, salts, and pesticide residues can differ between these different non-GM maize types in the non-GM reference groups.

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The introduction of unnecessary sources of variability and a significantly higher number of rats

fed a normal non-GM diet (320) compared to the GM-fed groups (80) per transformation event unbalances the experimental design. It would have been better to have a control group consisting of the same number of animals fed a mixture of these test diets. Furthermore, there is no provided data to prove that the diets given to the control and reference groups were indeed free from GM feed. According to European Union Directive CE/2001/18, the raw biochemical data required for statistical re-evaluation should be publicly available, but this is not always the case. In this instance, we had to obtain the necessary data through court actions against Monsanto for the MON 863 feeding study material (June 2005), or with the help of governments or Greenpeace lawyers. We would like to express our gratitude to the Swedish Board of Agriculture for releasing the NK 603 data, upon request from Greenpeace Denmark, and to lawyers from Greenpeace Germany for providing the MON 810 material on November 8, 2006.

This is the first time we have been able to directly compare data from three feeding trials with GMOs. We examined approximately 80 different biochemical and weight parameters in serum and urine after 5 and 14 weeks of feeding. These parameters were categorized based on their association with specific organs. The organs weighed at the end of the experiment, including adrenal glands, brain, gonads, heart, kidneys, liver, and spleen along with the whole body. We also analyzed parameters related to bone marrow and pancreas function. However, due to technical limitations or unknown reasons, some important measurements for evaluating liver function were not conducted. These measurements include gamma glutamyl transferase levels after

90 days of feeding as well as cholesterol and triglyceride levels in the NK 603 and MON 810 trials. Additionally, cytochrome P450 family members in all cases were not assessed. Furthermore, markers for sex differences such as blood sex or pituitary hormone levels were disregarded.

In accordance with OECD guidelines, it is well known that measurements should be conducted for at least 3 different experimental points to study dose or time-related effects. However, all three studies for the three GMOs only measured 2 doses and periods of feeding, without stating the reasons. This lack of data makes it difficult to evaluate dose and cumulative effects. In the annexes (Tables B, C, D), we have indicated the missing values for different parameters. It is important to note that the sample size of 10 for biochemical parameters measured twice in a period of 90 days is not sufficient to ensure a reliable statistical analysis presented by Monsanto. For instance, in a t test at 5% significance level, comparing 2 samples of 10 rats, there is a 44% chance of missing a significant effect of 1 standard deviation (SD), resulting in a power of only 56%.

In this case, the requirement for a power of 80% would result in a sample size of 17 rats. Thus, the statistical power is not adequate in these studies to dismiss all significant effects beforehand. This is particularly true for the magnitude of effects that typically manifest within three months, even though usual chronic toxicity may arise after a year of treatment. Consequently, the failure to reject the null hypothesis at a 5% significance level does not imply its truth.

Assessing statistical power is necessary

to understand undetectable size effects. The power depends on the sample size, effect size, and test level. An example of this is Monsanto's one-way analysis of variance (ANOVA) calculations. With a sample size of 10 animals for 10 groups and a 5% test level, there is a 70% probability of not detecting a medium size effect (e.g., 0.5 SD for a t test). However, within 90 days, chronic toxicity is more likely to produce medium rather than large size effects. It is crucial to improve the protocol at this stage. Based on Monsanto's analysis, it fails to prove the safety of consuming GM maize feeds. Any toxicity signs should be acknowledged to extend the experiment or reconsider the statistical analysis. A valid physiological interpretation of disturbed functional parameters per organ basis is the ultimate objective of this investigation.

In their report containing raw data and statistical analysis, Monsanto did not utilize their chosen and described statistical methods. Instead, they only employed parametric tests such as one-way ANOVA under the homoscedasticity hypothesis and Student t tests on contrasts. Furthermore, they only compared the data sets from the 33% GM maize feeding groups (for NK 603 and MON 810) with all reference groups in order to determine significant results. Their biological interpretation of these statistically significant results varied from case to case. For instance, they frequently used sex differences to dismiss any pathological significance, despite the absence of measurement of effects on sex hormone levels.

According to the study, the diet is considered safe, similar to what was suggested for MON 863 GM maize. However, there were slight deviations in the methodology used in the MON 863 experiments. In

those experiments, statistical significance was determined by comparing the 33% GM feeding dose group with the controls using ANOVA and contrast analysis. Significance was only established if the mean of the 33% GM feeding group fell outside the range of the mean of the reference cohorts. This increases the risk of obtaining false negative results. Due to inadequate statistical power to refute toxic effects observed in this study, there is concern about people and animals consuming these products before proper safety evaluations were conducted in vivo. To address this concern, we have employed an appropriate and experimentally validated statistical analytical methodology which will be further explained below.

The statistical methods used in this study were similar to those employed by Monsanto. We conducted the statistical analysis again to verify the descriptive statistics (such as sample size, means, and standard deviation) and ANOVA for each sex, variable, and GMO. The normality of the residuals was assessed using the Shapiro test, while the homogeneity of variances was evaluated using the Bartlett test. If both tests did not yield significant results, an ANOVA was performed. In cases where heteroscedasticity was present, we utilized the approximate Welch method. The Kruskal-Wallis rank sum test was selected when significance was observed in the Shapiro test.

To investigate how GM maize varieties affect each sex and diet, we compared the parameters of rats fed GM with control groups. Additionally, we compared these parameters with unrelated non-GM maize reference groups. To accurately evaluate individual effects of different normal diets, we calculated statistical differences between reference and control groups that took into account variations in salt, sugar,mineral,vitamin,and pesticide compositions. A suitable two-tailed comparison test was used

considering both normality (Shapiro test) and variance equality (F-test).

Based on the results, we conducted various statistical tests including an unpaired t test, a Welch corrected t test, or a Mann-Whitney test (for sample size of 10). To compare multiple pairs, we utilized the False Discovery Rate approach (FDR) to calculate adjusted p-values, aiming to control the false positive rate at 5%. Instead of employing Benjamini and Hochberg's method, we preferred using Benjamini and Yekutieli's method due to the dependencies among the parameters under examination. Furthermore, after normalizing the data, Principal Components Analysis (PCA) was performed to analyze the distribution of different factors such as sex, period, diet, dose, and group. Finally, we established representations and conducted paired tests for each rat and parameter within each group to explore temporal changes between the two feeding periods. All statistical computations were carried out using R language version 2.5 and involved specific packages: pwr package for power studies, bioconductor's multtest package for FDR, and ADE4 package for multivariate analysis.

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The negative perception of "Genetically-Modified" by the general public stems from concerns that GMOs may lead to more complex diseases. Previous studies discovered signs of toxicity in rats after they consumed MON 863 GM maize for a period of 90 days. However, it is important to note that these signs alone do not prove any

adverse effects on health. To further investigate, we examined feeding data from trials involving three different types of GM maize: MON 863, MON 810, and NK 603 (Tables 1,2; Annex Table E). By comparing our calculations with Monsanto's data (both significant and non-significant differences as shown in Annex Table E), we obtained ratios of 432/452 (NK 603), 435/450 (MON 810), and 442/470 (MON 863). Our statistical methods aligned with Monsanto's results (Annex Table E), but there were notable disparities in precision and interpretation of the primary outcomes. Therefore, we solely focused on analyzing relative differences above a threshold of 5% only (Tables I and II).

. 1. The results of the NK 603 feeding trials were initially analyzed. Table 1 presents the observations, including relative differences as compared to the control group. Out of the 23 effects that significantly differ and are believed to be caused by this genetically modified maize, 18 of them were observed in males (raw means with SEM; Annex Table F).

The distribution of effects varies according to sex. Liver (Fig. 1) and kidney (Fig. 2) parameters in all rats show differential expression between sexes. This is observed not only in the experiments involving NK 603 at week 14, but also at week 5 (data not shown). Similar observations were made in the feeding tests of MON 810 and MON 863 (Annex Fig. A- Fig.).

D). Male animals are more sensitive than female animals to physiological disturbances when fed NK 603. This is not seen in all three GM maize varieties. Additionally, most effects seem to be dependent on the dosage, as 83% of male effects only occur at the highest level of

GM maize concentration in the diet (33% feeding level). The greatest differences are observed in male kidney parameters, particularly urine phosphorus. This disturbance is dose-dependent and consistent over both 5 and 14 week periods of feeding.

The effects observed at this level are not likely to be a false positive result (week 5, 33%, adjusted p;0.003 for FDR calculated according to Benjamini and Yekutieli), given that all parameters were not independent. Similar results were also found for relative differences in lymphocyte and neutrophil levels (all for males, week 14, 33%, adjusted p;0.005). Table 1 shows the differences between NK 603-fed rats and controls.

The study examines the effects of GMOs, represented by percentage differences for each parameter compared to the control group, categorized by sex and dose. The results highlight significant differences compared to the controls (*p ;lt; 0. 05, **p ;lt; 0. 01) for all parameters evaluated in the subchronic feeding tests.

The parameters were categorized based on the organs they belong to, either by their synthesis sites or classical indicators of dysfunction. They were recorded for all groups if they displayed a significant ± 5% difference from the mean for at least one sex or one diet. The animals consisted of young adult rats, either male (m) or female (f), who were fed with the GM maize NK 603 (at 11% or 33% in the diet) for either 5 or 14 weeks. These rats were then compared to controls who were fed with a genetically similar maize line. The parameters were measured for 10 rats, except for the organ weights which were only obtained at the end of the experiment and involved 20 rats.

In the highlighted numbers, we indicate the statistical differences between GMO-fed rats and controls, which were not observed between the mean of the six reference groups and controls. A disparity between reference and control groups might suggest an effect solely from the diet itself.

The double-boxed numbers indicate the effects resulting from the GMO. These effects involve statistical differences between the GMO groups and the average of the six reference groups. The control and GMO treated groups did not consume genetically linked maize varieties. For the indicated parameters, differences are not significant according to a nonparametric test but are significant according to a parametric test. All other differences are significant according to both tests. "Lar Uni Cell" refers to the percentage of large unnucleated cell count. Fig 1 displays the Principal Component Analysis for liver parameters of all rats in the NK 603 feeding trial. The analysis at week 14 explains 66.65% of the total data variability (inertia) using 2 axes (49).

84% for factor 1 and 16.81% for factor 2, scaled d=2. This indicates the distinct differentiation of parameter values based on sex. Fig 2 presents a Principal Component Analysis for kidney parameters of all rats in the NK 603 experiment.

The obtained scheme for parameters at week 14 explains 44.78% of the total data variability (inertia) expressed on 2 axes (27.7% for factor 1; 17.51% for factor 2), with a scale d=2. This demonstrates a clear separation of parameter values based on sex. Out of the 18 GM maize-related effects compared to controls, 11 indicate that the groups of reference and control animals are similar in these cases (Table 1, framed values).

However, there are also significant

effects linked to GM (genetically modified) ingredients in comparison to all reference groups (Table 1, double framed values). At week 5, the most noticeable effects include a decrease in blood and an increase in urine creatinine clearance, as well as a decrease in blood urea nitrogen. However, these effects are not observed at week 14 (Fig. 3a,b). Despite this, the kidney parameters measured show the highest reactivity in both males and females, accounting for 52% of the significant effects observed. However, kidney parameters only make up 31% of all the measurements taken. Additionally, it is observed that ion concentrations are elevated in the urine of male rats fed GM maize. Furthermore, the weights of the liver and heart are also affected at the highest level of GM maize feeding (33%), with both organs showing up to an 11% increase in weight compared to corresponding parameters.

Variations in females are much less common (5/23) and do not show any significant differences except for urine phosphorus (major relative difference compared to controls) and blood potassium (compared to all groups). 3. 2. MON 810 Feeding MON 810 resulted in significant effects in females (11/15) (Table 2, crude means with SEM; Annex Table G), further emphasizing the differential effects between sexes. The observed sex-dependency for the measured parameters in the liver and kidney is applicable to all rats (Annex Fig. A ;amp; Fig.

B). The notable effects associated with GM-maize are typically observed after 14 weeks of consumption or when a high dose of GM feed is included in the diet. The affected parameters include blood cells, adrenal gland and kidney weights, an increase in blood urea nitrogen, and an elevated spleen weight.

In males, the most significant disruptions occur in liver function when the diet includes 33% GM-maize, leading to a slight decrease in general serum albumin production. All disturbances are less than 20% and the p-values indicate significance but are greater than 1%. However, when p-values are adjusted for FDR, they are not significant.

Table 2 shows the disparities between rats fed with MON 810 and the control group. For further information, refer to the explanatory notes provided in Table 1. 3.

We have previously discussed the evaluation of the rat feeding studies for MON 863 [5]. Sex-dependent differences are evident in the distribution of all parameters in the liver and kidney (see Annex Fig. C and Fig. D). Out of the 34 significant effects linked to genetically modified organisms (GMOs), 16 are found in males and 18 are found in females.

The results differ from those seen in NK 603 and MON 810. However, out of the males measured, 9 out of 16 (56%) had statistically significant differences in kidney function compared to only 4 out of 18 females. Although kidney measurements make up only 37. % of all data collected, these results indicate a gender-specific impact on kidney function.

In terms of liver parameters, there is a contrast between males and females. Males showed significant effects in 5 out of 16 cases, while females had a rate of 9 out of 18. The male rats also appear to be more susceptible to kidney disturbances at the higher GM feeding dose, with 11 effects at 33% compared to 5 at 11%.
There are additional statistically significant differences observed. In females, there is an increase in serum glucose and triglyceride

levels (up to 40%) compared to controls, as well as higher liver (7%) and overall body (3%) weight. Females also show elevated levels of creatinine, blood urea nitrogen, and urine chloride excretion. In males, there is greater variation in kidney function, including levels of creatinine, urine sodium, potassium, and phosphorus.
Furthermore, males experience a significant decrease (7%) in kidney weight and display noticeable chronic nephropathy [18]. There is also a decrease (3.3%) in male body weights and some differences in liver function (such as albumin, globulin, and alanine aminotransferase), though none of the FDR-adjusted p-values are considered significant.

Additionally, our study has measured the differences in time-related variations (at weeks 5 and 14) for this GM maize variety, comparing each feeding dose to controls. We have visually represented these variations for every rat and all parameters. Notably, the disturbed parameters are shown in figures 4-7. Our analysis clearly demonstrates that levels of triglycerides in female rats fluctuate between weeks 5 and 14 of feeding. Over time, triglyceride levels increase within the GM maize feeding group, while decreasing in the control group. Furthermore, in females, the increase in creatinine caused by MON 863 becomes more apparent with longer feeding periods, reaching an 11% level.

Another important difference (p=0.011) that we notice is a reverse change in female urine chloride excretion. In males, only urine potassium decreases over time when consuming GM feed, but it increases in controls (Fig. 7, p=0.

Overall, nearly all rats in the GM-fed treatment groups exhibit a tendency for physiological disturbance. The physio-pathological profiles differ based on the dosage or gender. Figure 3 shows a kinetic plot for urine creatinine clearance in male rats fed

NK 603. The lines represent the variations in this parameter (ml/min/100 g body weight) between week 5 and 14 for each rat on a 33% GM maize feed level and the control group. The thick dotted line represents the mean variation.

Fig 4 shows the kinetic plot of triglyceride levels in female rats during the MON 863 feeding trial. The variations in this parameter (mg/dL) between week 5 and 14 are represented by lines for each rat fed at the 11% GM maize feed level (a) and controls (b). The mean variation is indicated by the dotted thick line.
Fig 5 displays the kinetic plot of creatinine levels in female rats fed MON 863. Similar to Fig 4, the lines represent the variations in this parameter (mg/dL) between week 5 and 14 for each rat fed at the 11% GM maize feeding level (a) and controls (b). The mean variation is again represented by the dotted thick line.

The text above describes kinetic plots for urine chloride excretion in female rats fed MON 863 and for urine potassium in male rats fed MON 863. It indicates that for each rat in the experimental group (a) and the control group (b), the lines on the plots represent the variations in these parameters between week 5 and 14. The dotted thick line represents the mean variation.

The mean variation is represented by a dotted thick line. It should be noted that a "sign of toxicity" may only provoke a reaction, pathology, or poisoning, while a "toxic effect" is undoubtedly harmful in the short or long term. The statistically significant effects observed in this study for all three GM maize varieties

are indicative of toxicity rather than definitive proof. There are three main reasons for this conclusion. Firstly, the feeding trials were conducted only once and with only one mammalian species. It is recommended to repeat the experiments with multiple species of animals. Secondly, the duration of feeding was limited to a maximum of three months, which means that only acute and medium-term effects could be observed, if any, similar to those seen in processes like carcinogenesis or endocrine disruption in adults [19, 20].

Proof of toxicity is difficult to determine based on these conditions. It is important to conduct longer-term feeding experiments that last up to 2 years, as they are necessary to accurately assess the effects. This requirement is supported by the fact that certain health issues, such as cancer, nervous and immune system diseases, and reproductive disorders, may only become apparent after one or two years of intervention treatment. However, these effects may not be noticeable within three months of administration when initial signs of toxicity may be observed. Additionally, the current studies are limited by the number of animals used in each feeding group and the parameters studied, which may result in larger effects, such as a 40% increase in triglycerides, being missed. Furthermore, the low statistical power of the tests conducted is due to the experimental design of Monsanto (see Materials and Methods).

However, it is important to note that regulators rely solely on short-term (3-month) rat feeding trials to determine the safety of GM crop/food varieties compared to conventional types. Considering the widespread consumption of these GM crops by billions of people and animals globally, it is crucial to evaluate whether the

experimental design, statistical analyses, and interpretations used in these trials are adequate. Any differences observed in comparison to the isogenic variety should be considered as potential physiological disruptions. This is especially important because any statistically significant differences observed are unlikely to be due to population variation, as the rat strain used in these studies is genetically uniform. Additionally, the standardized conditions of rat maintenance, compliant with OECD standards, ensure that the diet is the only variable in the protocol. Therefore, the major distinction between treated rats and controls lies in the GM maize component of the test diet. The Tables indicate this difference with stars, representing the comprehensive physiological and pathological effects linked to GM. The framed results in the Tables emphasize that the effects from GM maize exceed those seen with any of the six different diets.The use of experimental diets containing high levels of salt or sugar could have been avoided if the focus of the research was solely on the overall toxicity of GM products. These diets led to higher levels of toxicity compared to other diets used during the 3-month feeding period, as observed.

In our study, we found a good overall agreement between our data and the results of Monsanto's original confidential reports. This was particularly true for the proportion of statistically significant observations. However, our methodology revealed different effects that changed the interpretation of the experimental results. Unlike the initial comments on the data, our study thoroughly considered sex differences. We examined and acknowledged differences in how male and female rats reacted to the GM maize test diets, taking into account established knowledge in areas such as endocrinology, embryology, physiology,

enzymology, and hepatology. These insights demonstrated sex-specific physiological and pathological effects. Our findings fully confirmed that effects on kidney and liver parameters were sex-specific for all rats in all three studies analyzed. It would have been unusual for both sexes to experience an identical effect, especially one indicating strong or acute toxicity.

This is not the case here, as we have also taken into account important effects that are not related to time or dose. We have provided details of these effects in our results. There is evidence suggesting a linear dose dependency, as requested by Doull et al.