In an attempt to determine any toxic effects by MegaHydrin™ on a macro and cellular level, a series

of detailed scientific evaluations were performed. The experiments were primarily based on testing how

living cells and tissue respond to a concentration gradient of MegaHydrin™ and if any concentrations

induced apoptosis or necrosis (death) of the cells. MegaHydrin™ indicated no signs of toxicity to the

cells, tissue membranes, organelle function and in no way induced a cytotoxic environment, apoptosis or

necrosis. Three types of cells were used in the evaluation: human erythrocyte (red blood cells), mouse

hybridoma and hamster ovary cell lines. Both the mouse and hamster cells have been accepted in the

scientific community to mimic the behavior of human cells.

The study of MegaHydrin™ on cells was mainly intended to look for the generation of a toxic

cytoplasmic environment, generally indicated by cellular death, cell disruption or a cease in cellular

metabolic function. Cytoplasm refers to the liquid environment within a cell.

A cell can die by several methods including apoptosis and necrosis. Apoptosis is a more natural

method of programmed cell death indicated by loss of water in the cytoplasm, cellular shrinkage, then cell

death. Necrosis is a mode of cellular death induced by a buildup of toxins that cause disruption in the

plasma membranes of the cell, cytoplasmic swelling and bulging of the cell, then finally the bursting and

death of the cell.

Looking at these two cellular mechanisms was the first step in

the determination of the safety of MegaHydrin™ in the body. A

series of solutions of MegaHydrin™ were prepared in water,

(milliliter) to a supersaturated solution of grams per mL. Each of

these MegaHydrin™ preparations were introduced into separate

vials containing live cells of each of the three cell types. The initial

reaction with the cells was observed, then every 15 minutes for over

six hours. At each observation, the MegaHydrin™ treated cells

were compared with live cells that were non-treated, which acted as

a control. Each observation included microscopically looking for

any cell disruption, bulging, shrinkage, etc. At each observation, the

cellular concentration was also measured using a device called a

hemocytometer.

MegaHydrin™, in every tested concentration, showed no signs of inducing necrosis or apoptosis over

the course of the experiment to either the erythrocyte, hybridoma or the ovary cells. In fact, watching the

Red blood cells were one type of cell

tested that indicated no signs of toxicity

with MegaHydrin™ treatment.

cells over the course of the experiment, more cells (up to 50%) more were living in the MegaHydrin™

treated vials than in the non-treated controls.

In every living cell there are specific enzymatic activities that are constantly occurring that indicate the

health state of a cell. If these enzymatic reactions cease, then the cell will die or otherwise be considered

cytotoxic. The esterase activity may cease through the introduction of toxins into the cell cytoplasm or

through the permeation or degradation of internal membranes. The use of two chemicals, calcein AM and

ethidium homodimer-1 are used extensively to test for cytotoxicity and without question, objectively

determine whether a cell is live and healthy, or dead and toxic. The idea

behind this assay is that there is a specific enzyme, known as esterase

that only is active within a healthy cell. Calcein AM binds with the

products of the intracellular esterase activity and produces a bight green

fluorescent color. If the esterase activity ceases, there will be no green

fluorescence. In the event that any membranes break down, or the

integrity of the cell is otherwise jeopardized, then the ethidium

homodimer-1 reacts with the nucleic acids to produce a vibrant red

fluorescent color. Cells may be monitored simultaneously for both

fluorescent types.

The basic assay is very easy. If the cells are green, they are healthy.

If they are red, they are dead. On a more advanced level, a device

known as a spectrofluorimeter may be used to calculate the exact

intensity of the fluorescence to definitively illustrate the exact amount of

cells live and dead. Similar to Test #1, this assay used a gradient of

MegaHydrin™ treated cells that had been exposed, or stained, with the calcein AM and ethidium

homodimer-1. After 30 minute increments, the fluorescence intensity was measured for both the treated

cells and non-treated controls.

Again, MegaHydrin™ treated cells, in any concentration, showed no signs of cytotoxicity or cellular

death. Interestingly enough, in this assay as well, more cells were healthy in any arbitrary observation

than the non-treated controls.

Every cell is made up of microscopic components known as organelles. Some organelle types include

mitochondria, liposomes, nuclei, etc. Each type of cell and cellular component has small electric charges

intrinsic to that component, some being positive while others’ negative. Because of these small charges

on these molecules, a technique called electrophoresis can be used to evaluate the cells and organelles.

Electrophoresis involves using an electric field to analyze charged molecules. Because each cell and

organelle type as its own intrinsic charge, they have predictable characteristics when they are analyzed.

Under a given electric field, a cell should repeatedly travel the same distance over a given time under

analysis. This constant ratio of the distance and time to the electric field is known as its electrophoretic

mobility. Each cell or organelle type has its own distinct electrophoretic mobility.

Using the principles of electrophoresis in a device called a CE-LIF, Capillary Electrophoresis with

Laser Induced Fluorescence, MegaHydrin™ treated cells were analyzed for toxicity. There are numerous

fluorescent compounds to place a fluorescent label on different organelles. In this assay, three dyes were

employed to mark mitochondria, lysosomes and whole cells. With a "marked" cell or organelle, a

fluorescence detector in the CE-LIF can measure the electrophoretic mobility of that molecule. Any

change in the health of the cell, such as induced toxicity, apoptosis, necrosis, etc., will change the

electrophoretic mobility through changing the overall net charge on the cell.

Solutions of MegaHydrin™ in a gradient of concentrations were used added to cells and organelle

preparations stained with an appropriate dye. The electrophoretic mobilities of the MegaHydrin™ treated

cells were compared to that of the non-treated cells.

In all of the assays, the measured electrophoretic mobility of the MegaHydrin™ treated cells and

organelles did not differ from the non-treated, indicating that no internal change had been made to the cell

or cellular component. This lack of differentiation illustrates that the cellular components of the

MegaHydrin™ treated cells were as healthy as the control cells, showing no signs of cytotoxicity.

Each of the three main toxicity tests employed with the investigation of MegaHydrin’s™ potential

toxic effects on cells, distinctly conveyed NO INDUCED CELLULAR TOXICITY. Not only has the

research enlightened scientists on the safety of MegaHydrin™, but also introduced new and profound

properties of MegaHydrin™, such as the increase in the lifespan of the cells, that have since been further

studied by the Flantech Group.

Make sure to check out our information about the scientific studies on MegaHydrin™ as an effective

and efficient antioxidant, proven to work against hydroxyl and superoxide free radicals in addition to

singlet oxygen and other reactive oxygen species.

We invite your questions and comments:

271 Airpark Rd.

Cottonwood, AZ 86326

USA

Phone: (928) 634-2668

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