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Male mice fed doses of aspartame far lower than the U.S. Food and Drug Administration (FDA) recommends got slower and dumber working through a maze — a literal and metaphorical test of human intelligence since ancient times, before Homer wrote of Theseus chasing the Minotaur through the mythic labyrinth in the “Iliad.”

According to a study published Aug. 31 in Scientific Reports, the mice passed on the learning and memory deficits to their first-generation offspring — but not second-generation offspring.

The researchers studied males and not females because they were looking for problems caused only by genes and not by direct exposure. Fetuses developing inside an exposed female would experience both.

Aspartame, an artificial sweetener found in more than 6,000 foods and medicines, has been linked to heart disease, obesity, mood disorders and other serious health issues. In the U.S., it’s sold under the brand names Equal and Nutrasweet.

Researchers led by Pradeep Bhide, Ph.D., at the Florida State University College of Medicine, provided adult 8-week-old male mice with free access to drinking water containing either 0.015% or 0.03% (by weight) of aspartame.

A third group of control mice drank plain water.

“A key public health implication of our findings,” the authors wrote, “is that the population at risk of aspartame’s adverse effects on learning and memory may be larger than current estimates, which consider the directly exposed individuals only. Our findings underscore the need for considering heritable effects as part of the safety evaluation of artificial sweeteners by regulatory agencies.”

Mice in the 0.015% and 0.03% aspartame groups consumed, on average, 43.2 milligrams and 86.4 milligrams of aspartame per kilogram of body weight per day.

“Milligrams per kilogram” (mg/kg), is a way to express dosages relative to a subject’s weight, to account for a larger subject requiring a larger dose to obtain a specific effect.

The FDA-recommended maximum daily intake value of aspartame for humans is 50 mg/kg but most consumers take in much less — 4.1 mg/kg.

Mice in the lower-concentration aspartame group received approximately this dose, while those in the 0.030% group got about twice the average daily intake. So low and high dosages were just 8.2% and 16.4% of the maximum “safe” agency-recommended intake, respectively.

During the 16-week experiment, all mouse groups underwent tests for spatial working memory, spatial learning, reversal learning and learned helplessness, which are used to assess the cognitive or learning effects of drugs, foods, or other interventions.

Bihde’s study design was typical for mouse experiments except for his focus on male mice and their male or female offspring. Most studies examining the heritability of characteristics use female study animals.

He selected this mouse strain because a previous study showed the animals neither preferred nor avoided aspartame in drinking water, nor did they experience changes in weight or metabolism after exposure.

Aspartame recently was in the news as a possible cancer-causing agent. Based on what it termed “limited evidence” the World Health Organization considers the sweetener, at a maximum daily exposure limit of 40 mg/kg, as “possibly carcinogenic [cancer-causing] to humans.” WHO does not recommend using artificial sweeteners for weight control.

U.S. regulators disagree with the WHO’s position and even recommend a higher daily maximum intake.

As The Defender reported last week, industry representatives are paying dietary influencers on social media to promote artificially and naturally sweetened products to children.

Testing: How did the mice perform?

Mice in all three dosage groups underwent regular cognitive testing during the study. Researchers found deficits in spatial working memory in treated mice relative to controls at 4 weeks, an effect that persisted at weeks 8 and 12.

No differences between high- and low-dose groups were evident.

The spatial learning and memory test used a maze to determine how long it took mice to find a way out and the number of mistakes they made before escaping. This test began at 14 weeks after initial dosing and continued for 10 consecutive days.

Mice typically improved over time on this test, which was true for all three groups.

But mice ingesting aspartame found their way out of the maze much more slowly than control animals. Again, no difference was noticed between treatment groups.

Learned helplessness,” a term from human psychology, describes a feeling of being stuck in a situation or circumstance and being “paralyzed” from acting.

Psychologists use learned helplessness tests to characterize depressive episodes. Animals undergoing learned helplessness testing are observed for how hard they try to avoid an apparently inescapable bad situation, for example, an electric shock.

To assess learned helplessness Bhide and co-authors used a tail suspension test, which involves hanging mice upside down by their tails to quantify the effort they expend in pulling up and righting themselves. They found no difference here between aspartame and control groups.

Cognitive dysfunction: the next generations

Treated and control mice were bred with stock females to produce second-generation test animals. Litters were of normal size, with pups meeting normal developmental milestones. However, several deficits noted in the original test mice were also seen in these unexposed animals.

The effects on spatial working memory for treated vs. control fathers were particularly pronounced. Offspring of both dosage groups also showed significant learning deficits compared to the control lineage.

But no differences were apparent between offspring of low- and high-dose aspartame fathers, or for reversal learning — a measure of how an animal unlearns old, ineffective behavior and develops new problem-solving strategies.

Deficits among first-generation mice in learned helplessness were also not evident.

To eliminate the question of whether these effects are a permanent part of the animals’ DNA, vs. a temporary effect on sperm cells, researchers bred first-generation males to produce second-generation litters.

To simplify their analysis they compared learning test responses of second-generation control group offspring only to the second-generation higher aspartame dosage group but found no second-generation effects.

Why just male mice?

Bhide’s focus on paternal lineage is unusual for inter-generational toxin exposure studies. Historically, most investigations consider only maternal exposures, particularly on events occurring during pregnancy or nursing.

Since a father’s biological involvement ends at conception, intergenerational effects must occur through effects on exposed males’ sperm cells. If these effects had been permanent, second-generation mice as well as first-generation offspring would show learning deficits, but this was not observed.

Until relatively recently, biologists believed that acquired characteristics were not heritable. While this remains true for most traits, scientists now recognize the potential for certain drug, food or toxic exposures to turn genes on or off temporarily.

Epigenetics is the emerging science describing how certain life events, including toxic exposures, may act as temporary genetic switches.

Epigenetics also explains how a drug or pesticide might cause harm to individuals with no history of exposure, and how this effect eventually disappears.

The alternative — permanent genetic damage — would continue to affect offspring for generations. The effects of aspartame lasted only one generation, which is consistent with transient, reversible epigenetic alterations to sperm cells.

Considering epigenetic effects, and not just direct contact with toxins, amplifies the potential harms of certain exposures and expands the scope of potential consequences to exposure that regulators should consider before licensing or approving certain products.