geranyl acetate

Three related compounds

Geranyl acetate and geraniol good medicines or benign food additives?

It’s impossible to comment on the former in a manner that the FDA would approve. This post will compare a study showing that geranyl acetate and geraniol induce apoptosis in a cell culture model with rodent toxicology tests.  The conclusion is of the first study is that geranyl acetate has potential as a chemotherapy agent.  Chemotherapy agents are often mutagenic. 

The first study will be interwoven with a long term rodent study examining potential carcinogenic effects. Geranyl acetate is after all a food additive.

Is geranyl acetate a safe food additive?

The second study was conducted by U.S. Department of Health and Human Services, the National Institutes of Health. The document is a rather extensive rodent study examining both short term and long term effects with an emphasis on carcinogenesis. It was published in 1987 Geranyl acetate was tested because of its use in foods and because it had not previously been tested for carcinogenicity. Human exposure to this flavoring agent occurs through foods, skin care products, and perfumes. Injection into the stomach, gavage, was chosen over addition to feed because of geranyl acetate’s volatility and its reaction with moisture in feed. This post skips over short and medium term sub studies of the major study. Both these strains are prone to spontaneous tumors.

Rats and mice were observed twice daily for mortality and were weighed weekly. Necropsies were performed on all animals.

2 year studies

• 50  F344/N rats of each sex  received 1,000 or 2,000 mg/ kg body weight
• 50 B6C3F1 mice of each sex received 500 or 1,000 mg/kg geranyl acetate in corn oil by gavage, 5 days per week for 103 weeks. 
• Vehicle controls received corn oil alone.

All animals were observed twice daily for signs of morbidity or mortality. Major tissues or organs were examined for grossly visible lesions. 

Cell and rat viability

The MTT assay measures viability and redox balance. Colo-205 cancer cells were grown overnight. Fresh medium with geraniol and geranyl acetate separately at different concentrations (0-100 μM) for 24 hrs. A 0.5 mg/ml MTT solution was added for the last 4 hours of incubation.  The absorbance was measured at 570 nm.  The molecular mass of 196.3 g/mole.  100 µM is about 0.02 g per liter.  .A liter of water weighs 1 kg.  Note that this does is far less than what the rats received in some ways of looking at things.

Cells

rats

One might argue that reducing equivalents measured by the MTT assay may contribute to growth of cells in culture or whole rats.

Programmed cell death (Apoptosis) and whole animal death

Colon cancer colo-205 cells were treated with 20 μM geraniol or 30 μM geranyl acetate for 24 hrs. cells were stained with DAPI, a fluorescent DNA dye that will stain dead and live cells.. When DAPI binds to DNA it emits blue light when excited with ultraviolet light. Under normal circumstances

The apoptotic cells show more intense DAPI staining suggestive of fragmentation of the tightly coiled chomosomes increasing the access to the dye. Rodent is tracked by a Kaplan and Meier curve is basically a tracker of animals dropping off the chart of the living.

After 30 weeks most of the rats are still alive, i.e. probability of survival is close to 1.0 or 100%.  By 90 weeks only ~67% of the male rats on the 2g/kg high dose are still alive. In male rats, the survival of the high dose group was significantly less than that of either the controls (P=0,00l)or the low dose group (P=0.003). The survival of the high dose group of female mice was significantly less than that in the control or low dose groups (P<0.001). The survival of the low dose group was significantly less than that of the controls (P=0.020). No significant differences were observed between any groups of male mice. [2]

DNA Damage and outright cancer

Comet assay to detect DNA damage

1. Cells were harvested in phosphate buffered saline and embedded in a gel like substance called agarose.
2. When the agarose had solidified the cells were lysed.
3. Electrophoresis buffer was added. An electric potential difference was applied. DNA being anionic (negatively charged) migrated to the anode.

The bigger the tail, the more DNA damage.

Pheochromocytoma adrenal tumors occurred in male mice with a positive trend p=0.03

Pituitary: Adenomas were seen in male rats with a negative trend (P<0.02; control, 10149, 20%; low dose, 8/50, 16%; high dose, 2/48,4%). In pairwise comparisons between control and dosed groups, the incidence in the high dose group was lower (P<0.02) than in the controls. Results of the life table analyses of adenomas in male rats were not significant. This tumor was observed in 13/47, 16/43, and 9/48 female rats.

Pancreatic islet-cell adenomas or carcinomas (combined) were observed in male rats

• control, 4/49, 8%
• low dose, 3/48, 6%
• high dose, 0/50,0%,

The study authors did not consider this decrease significant when overall survival was considered. Results of the pairwise comparisons between control and dosed groups were not significant, and these tumors were not observed in female rats.

Hematopoietic System: Malignant lympho- mas were observed in male mice with a negative trend (P=0.018) and the incidence in the high dose group was lower (P=0.044) than in the

• controls7/50,14%
• low dose, 2/50,4%
• high dose, 1/50, 2%.

Malignant lymphomas (mixed type) in male mice were observed less in the  (P=0.041)

• control 3/50, 6%
• low dose 0/50, 0%
• high dose 0/50,0%.

In female mice, these tumors were not observed in statistically significant proportions.

Thyroid: Follicular-cell adenomas

Skin cancers in male rats. Squamous cell papillomas in the skin were increased marginally in low dose male rats

• control, 0/50
• low dose, 4/50, 8%, one also had a  squamous cell carcinoma
• high dose, 1 /50, 2%

The incidence of low dose male rats with either squamous cell papillomas or carcinomas was greater (P<0.05) in comparison with the controls.  All of these tumors were found during weeks 103 and 104. In female rats, these tumors were not observed in significant proportions.

Thyroid: Follicular-cell adenomas were observed with a negative trend (P=0.024, Cochran- Armitage) in female mice, and the results of the pairwise comparisons between the control and high dose groups were significant (P=0.030, Fisher): control, 5/50,10%; low dose, 3/48,6%; high dose, 0149, 0%. This decrease was not significant when survival differences were taken into account. This tumor was not observed in significant proportions in male mice.

References

1. Qi F, Yan Q, Zheng Z, Liu J, Chen Y, Zhang G. (2018)Geraniol and geranyl acetate induce potent anticancer effects in colon cancer Colo-205 cells by inducing apoptosis, DNA damage and cell cycle arrest. J BUON. 2018 Mar-Apr;23(2):346-352. free article
2. National Toxicology Program. NTP Carcinogenesis Studies of Food Grade Geranyl Acetate (71% Geranyl Acetate, 29% Citronellyl Acetate) (CAS No. 105-87-3) in F344/N Rats and B6C3F1 Mice (Gavage Study). Natl Toxicol Program Tech Rep Ser. 1987 Oct;252:1-162. free report