Reproductive toxicity of d-004 (a lipid extract from roystonea regia fruits) in rats

Toxicidad reproductiva del d-004 (un extracto lipídico de los frutos de roystonea regia) en ratas

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Benign prostatic hyperplasia (BPH), a progressive and non-malignant enlargement of the epithelial and stromal elements of the prostate gland.^1,2 Consequently, affected men often experience lower urinary tract symptoms (LUTS) that may impair considerably their quality of life.³ Observational studies have shown that the incidence of BPH in the ageing men has been increasing.^4,5,6Although BPH is a multifactorial process, that involves both hormonal and non-hormonal factors, there is wide evidence that hormonal changes occurring in the aging man plays a pivotal role in the pathogenesis of BPH. The increased conversion of testosterone in dihydrotestosterone (DHT), mediated by prostate 5α-reductase enzyme, is the main hormonal factor of BPH, since the accumulation of DHT promotes excessive prostatic cell growth, BPH also involves non-hormonal factors as the increased adrenergic tone of prostate smooth muscle, mediated through α1-adrenoceptors.^6,7,8

Consequently with the etiological factors leading to BPH/LUTS, the two major drugs classes to treat BPH are 5α-reductase inhibitors, like finasteride and dutasteride, and a-adrenoceptor blockers, like doxazosin, terazosin, tamsulosin and alfuzosin. The combined therapy with 5α-reductase inhibitors and α1-ADR blockers provides the benefits of both therapeutic classes.^9,10,11 D-004, a lipid extract obtained from the mature fruits of the royal palm by a process that includes a first alkaline hydrolysis and a further solvent (n/hexane) extraction, contains a mixture of free fatty acids (mainly oleic, lauric, palmitic and myristic acids) wherein oleic acid is the most abundant. D-004 has shown to inhibit the activity of rat prostate 5a-reductase in vitro,¹² and prevents prostate hyperplasia induced with testosterone.^13,14,15,16 Also, D-004 has demonstrated to antagonize α1-adrenoreceptors-mediated responses both in vitro and in vivo.^17,18Single oral dose toxicity of D-004 (2 000 mg/kg) in rats and rabbits has been practically absent, and the same was noted in the subchronic oral studies in rats and mice treated with doses up to 2 000 mg/kg for 90 and 60 d, respectively.^19,20 In addition, D-004 has not shown evidences of cytotoxic or genotoxic potential in tests able to detect the ability of chemicals to induce gene mutations (Ames test) or to provoke chromosomal aberrations in somatic cells (Bone marrow micronucleus test).^21,22 This study was conducted to provide information on the possible reproductive toxicity in Sprague Dawley rats when administered to fertile rats of both sexes.

No evidence of toxicity was observed during treatment. Mean body weights of mothers of parental generation in the treatment groups were not significantly different from the control group during gestation and lactation periods, but body weight gain in the group treated with D-004 1000 mg/kg/d was significantly less than control in the period between 0 to 6 days of gestation (Table 1). Treatment did not appear to affect food consumption (data not shown). One female in the 1 000 mg/kg group died due to gavage error. No apparent treatment- related signs of reproduction toxicity were associated with D-004 exposure. The length of gestation was unchanged by treatment, like fertility, gestation, viability and lactation indexes (Table 2).

Table 1.
Average maternal body weight gains changes (g) in D-004 rat oral reproductive toxicity study.

^a Mean ± standard deviation (number of dams); bp< 0.05, Significantly different from vehicle control (ANOVA).

Table 2.
Summary of reproduction data of F0 females in the D-004 reproductive study.

^a Mean ± standard deviation.

There were no differences among groups in the litter size, the number of pups born live or dead, the number of pups that survived until weaning and the pups body weights through the lactancy (Table 3). In this study a low neonatal mortality and no external abnormalities were observed. Dams and their offspring were healthy during lactation. No unusual maternal behaviour was observed. Drug-treated and control groups' offspring were comparable in physical and sensorial development and reproductive performance (Table 4). The general health and behavioural condition of offspring was good in all groups. There was no significant effect of treatment on the motor development, sensory function, open field activity and onset of reflexes (data not shown).

Table 3.
Viability and body weight of F1 offspring in the D-004 reproductive study.

^a Mean ± standard deviation (number of litters examined). PND (postnatal day).

Table 4.
Physical and reflex development on F1 pups (%).

^a (Positives/examined) x 100

The fertility of the F1 generation was comparable in the control group and those treated with D-004. In turn, no treatment-related effects were apparent on F1 generation reproductive parameters such as, fertility and gestation index, litter size and duration of gestation period. (Table 5). Mean body weights gains of F1 mothers in the treatment groups had no significant different from controls during gestation, and weight gain was similarly unaffected (Table 6). Treatment did not appear to affect maternal food intake neither (data not presented). There were no significant differences among groups of F2 generation regarding to number of pups born, number of pups born live or dead, mean pup’s weight and postnatal pups survival (Table 7). No offspring with malformations were found in any experimental group.

Table 5.
Summary of reproduction data of F1 females in the D-004 reproductive study.

^a Mean ± standard deviation (number of dams).

Table 6.
Average maternal body weight changes (g) during gestation of F1 females in the reproductive toxicity of D-004.

^a Mean ± standard deviation.

Table 7.
Viability and body weight of F2 offspring in the D-004 reproductive study.

^a Mean ± standard deviation (of litters number examined). PND (postnatal day).

In the present study we report the results of the evaluation of reproductive toxicity potential of oral treatment with D-004, which has shown to inhibit competitively prostate 5 α- reductase activity in vitro,¹² an effect attributable to free fatty acids present in D-004, like lauric, myristic and oleic acids,²⁹ and prevents prostate enlargement induced with T in rodents.^13,14,15,16 Likewise, D-004 has displayed in vivo and in vitro antagonism of α1- adrenoreceptors.³⁰ Drugs used to treat BPH have been associated with adverse effects, such as gastrointestinal disturbances (nausea, diarrhoea), impairment of male sexual activity (reduced libido, erectile dysfunction, abnormal ejaculation), gynecomastia, orthostatic hypotension, dizziness, headache, asthenia and rhinitis.^{7,31,32,33,34} In contrast, plant extracts used to manage BPH, have not been associated with drug-related adverse effects.³⁵ Saw palmetto, that is most commonly used, has been considered to produce fewer side effects than traditional medications,^36,37 but published experimental toxicity studies are limited.^38,39 The composition of D-004 partially resembles that of the lipid extract of saw palmetto (Serenoa repens) fruits.

D-004 is a lipid extract of royal palm (Roystonea regia) fruits that consists in a mixture of free fatty acids, wherein oleic, lauric, palmitic, and myristic are the most abundant. Animals and humans have consumed regularly the major individual fatty acids contained in D-004 (oleic, lauric, palmitic and myristic) from the food chain, it was rationale to believe that these compounds should not be toxic for humans. An old study referred the lack of oral single dose (15-19 g/kg of body weight) toxicity of oleic, lauric, palmitic, myristic, or stearic acids in rats.⁴⁰ No-observed-adverse-effect level values higher than > 6 000 and 5 000 mg/kg have been reported for lauric acid administered orally to male rats for 18 weeks for palmitic acid administered to rats for 150 d, respectively,⁴¹ while myristic acid did not induce a mutagenic response in either bacterial or mammalian systems in vitro, and that its use as food flavouring does not pose a health risk to humans.⁴²On the other hand, the regular intake of oleic acid with the diet, as occur in the Mediterranean diet has been associated with cardiovascular benefits rather with adverse effects.^43,44 These evidences supported that the intake of D-004 should not suppose a relevant safety risk for humans.

This study demonstrates that D-004 (500-1 000 mg/kg) induced no preweaning mortality, reduce body weight, developmental delay, or behavioural changes in the FI offspring. Maternal body weight gains in the group treated with D-004 (1 000 mg/kg/d), was less than control in the period between 0 to 6 days of gestation, but such a finding is not considered an indicator of maternal toxicity. Thus, this finding appears to be a random result, within the normal fluctuation of such a parameter in such a period and species. Considering this fact, joined to the absence of any other symptoms of toxicity related to D-004 treatment, we can expect that such effect could be out of biological relevance. In addition to, the weight of each pups in this group is on the range know for this species in our conditions in previous studies. Nevertheless, we will take into account this results in furthers studies, since was found at the higher doses tested.

It is concluded that oral treatment of rats of both sexes with D-004 (500 - 1 000 mg/kg/d) affected no the fertility and general reproductive function, which indicates that it does not alter the lactation, growth, development and sexual maturation of the progeny.