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Identification of dose-response relationship, sensitive target organs and specific biomarkers when testing PHA-E lectin in a 28-day rat study

This result comprised the 28-day feeding and toxicity studies, which formed the basis for the 90-day animal studies in the project.

The specific objectives of this task were to:
-Optimise the composition of the purified diet to include the maximum concentration of rice.

- Identify sensitive target organs and specific biomarkers for the identification of adverse effects of the PHA-E lectin in the rat model.

- Establish the lowest-observed-adverse-effect-levels (LOAEL) for the lectin, which are needed for the later 90-day study (Task VI).

In order to fulfil the objectives of the project, PHA-E lectin seemed to be the most useful model compounds. Consequently, the project partners decided that all efforts in making the recombinant proteins should concentrate on PHA-E lectin on behalf of the GNA lectin and Bt toxin.

The increased production of the PHA-E lectin resulted in a yield that was sufficient to perform all scheduled studies for this compound.

In the preliminary small-scale acute rat study the purpose was to reveal the toxic potential of the PHA-E lectin expressed in P. pastoris. Based on findings such as oedema and thickening of the epithelium of the small intestine and sign of proliferation in the gut lympoid tissue it was concluded that 10 mg/rat/day (corresponding to about 0.1% in the feed) would generate adverse effects in the animals. In order to avoid mortality and serious adverse effects that could compromise the evaluation of the study, a level of 0.08% PHA-E lectin in the diet was therefore chosen as the highest dose level in the 28-day study with PHA-E lectin.

Before initiating the animal studies one very important item was to test whether the rats liked the taste of raw brown rice and whether they could tolerate high inclusion levels of rice in the diet.

The results from the tolerance study showed that Wistar rats readily eat and tolerate up to 60% of raw brown rice in a purified diet for 28 days.

In the first 28-day study with PHA-E lectin focussing on general toxicity, no dose-related effects were seen on clinical observations, body weight, body weight gain, and food consumption. For clinically parameters as blood chemistry, haematology and microbiology, a few numbers of significant clinical signs were observed in the study period, but none of them are considered to be treatment-related. At the necropsy, animals given PHA-E lectin had a tendency to slightly thicker jejunum compared to animal from the control group. Despite these macroscopic observations, the findings could not be confirmed by histologic examinations. In general no dose-related findings in organ weights, macroscopic pathology and histopathology were observed.

The second 28-day study with PHA-E lectin focussed on immunotoxicity of the PHA-E lectin and especially designed to reveal this. Other end-points addressing general toxicity were included as well. In the study a statistically significant increase in absolute and relative weight of the small intestine of female rats and relative weight for male rats was observed in the highest dose group. In addition, the absolute and relative pancreas weight was significantly increased in nearly all female groups given PHA-E lectin compared to the control group.

To conclude on the two studies with PHA-E lectin, the toxicity of PHA-E lectin expressed in P. pastoris was lower than expected. Accordingly, a dose-response relationship for PHA-E could not be established based on these studies. An effect level of 0.08% PHA-E has been established based on the increased weight of the small intestine. As a consequence, the small intestine and related parameters and partly the pancreas will be the target organs for the future 90-day study with PHA-E. As the PHA-E level of 0.08% in the feed only caused mild effects, it was decided to increase the level of spiked PHA-E-lectin in the 90-day study with PHA-E rice to 0.1%. Based on the findings in the acute study we had expected an effect of the PHA-E lectin at a lower dose level than 0.08%. However, the rats seemed to in some way have been adapted to the effects of the PHA-E lectin indicating that the effects were acute and reversible of nature.

Nevertheless, agglutination assays indicated that PHA-E lectin had very similar biological activity to authentic plant standards. The glycosylation of recombinant PHA-E was typical of that previously seen for Pichia pastoris produced proteins.

The special formulated purified diet with 60% of rice tested in the preliminary tolerance study as well in the two above-mentioned 28-day studies proved to sustain growth and health and yield reproducible experimental results. This type of diet was therefore with only minor corrections the diet that was used in the 90-day studies.

More information on the project can be found at:

Informations connexes

Reported by

Danish Institute for Food and Veterinary Research
Morkhoj Bygade 19
2860 Soborg
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