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Transgenerational hormonal imprinting caused by vitamin A and vitamin D treatment of newborn rats. Alterations in the biogenic amine contents of the adult brain
Brain & Development 31 (2009) 666–670 www.elsevier.com/locate/braindev
Original article
Transgenerational hormonal imprinting caused by vitamin A and vitamin D treatment of newborn rats. Alterations in the biogenic amine contents of the adult brain Korne´lia Tekes a, Melinda Gyenge a, Mo´nika Hantos b, Gyo¨rgy Csaba c,* a Department of Pharmacodynamics, Semmelweis University, H-1445 Budapest, Hungary Department of Pharmacy Administration, Semmelweis University, H-1445 Budapest, Hungary c Department of Genetics, Cell and Immunobiology, Semmelweis University, P.O. Box 370, H-1445 Budapest, Hungary b
Received 16 June 2008; received in revised form 22 October 2008; accepted 23 October 2008
Abstract Biogenic amines (norepinephrine, dopamine, homovanillic acid, serotonin and 5-hyroxyindole acetic acid) were measured by HPLC method in adult F1 generation rats’ brain regions (brainstem, hypothalamus, hippocampus, striatum and frontal cortex), whose mothers (P generation) were treated with vitamin A or vitamin D neonatally (hormonal imprinting). Many significant differences were found, related to the maternally untreated controls. In the earlier studied P generation females, vitamin A consistently influenced the serotonerg system (5HIAA), while vitamin D the dopaminerg system (DA or HVA). Vitamin A imprinting always resulted in reduced, while that by vitamin D always in increased tissue levels. In the present case (directly untreated F1 generation) the transgenerational effect was not unidirectional, however biogenic amine tissue levels were strongly disturbed and brain-area dependent. The results call attention to the transgenerational effect of hormonal imprinting in the case of receptor level acting vitamins which are frequently used in the most imprinting-sensitive period (perinatally) of human life and suggests that caution is warranted. Ó 2008 Elsevier B.V. All rights reserved. Keywords: Neonatal treatment; Transgenerational effect; Vitamins; Biogenic amines; Imprinting
1. Introduction Hormonal imprinting is a well-established phenomenon, that takes place in the developing cells when a hormone meets its target receptor. Usually, it occurs in the perinatal period, but it can happen later as well, when the target cell is in a developing phase [1–3]. As a result of imprinting the binding capacity of the receptor is modulated reaching its value characteristic for the adult animal [4]. Without imprinting there is no normal receptor maturation [5] however, the over-abundance of the target hormone or even foreign molecules that are able *
Corresponding author. Tel./fax: +36 1 210 2950. E-mail address: [email protected] (G. Csaba).
0387-7604/$ - see front matter Ó 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.braindev.2008.10.007
to bind to the receptor, can cause ‘‘faulty imprinting” [1–4] with life-long genetic, morphological, biochemical (alteration of cell proliferation, change in hormone production) and behavioral (loss of libido, aggressivity) consequences [6–14]. The effect of imprinting is not limited on the direct targets but – by a presently unknown mechanism – it can also have effect on other members of the endocrine system [15–17]. The consequences of imprinting can be transmitted to the progeny generations as well, and even in the F1 and F2 could be observed [18–20]. The behavioral effects of imprinting called our attention to the possibility of the influence of imprinting to brain targets, which has been suggested by many previous experiments [21].
K. Tekes et al. / Brain & Development 31 (2009) 666–670
Previously, we studied the imprinting effect of vitamins A and D, two molecules that act by binding to receptors belonging to the steroid receptor superfamily. Both compounds are used widely during the perinatal critical period as preventive treatments. The effect of these compounds on the biogenic amine neurotransmitter levels in the brain was demonstrated [22]. Considering these results, the present experiment was addressed to the transgenerational imprinting effect by these vitamins on the biogenic amine tissue levels in the brain. 2. Materials and methods 2.1. Animals and treatment Female newborn (strictly before the age of 24 h) Charles River originated Wistar rats (P generation) of our closed breeding colony were treated with a single dose of 0.05 mg vitamin D3 (cholecalciferol, Sigma, USA) or with 3 mg vitamin A (retinol, EGIS Budapest, Hungary) solved in 0.1 ml sunflower seed oil. Compounds were given subcutaneously under the skin of the neck. Controls got the vehicle. The animals were housed at room temperature under normal light cycle. Food and water were available ad libidum. When the animals were 3 months old they were mated with control (untreated) males. Two months old female progenies (F1 generation) were used for the experiments. All of the animal experiments were executed according to the Council Directive No. 86/609/EEC. Animals under ether anesthesia were sacrificed by decapitation, brains immediately removed and five regions (frontal cortex, hypothalamus, hippocampus, striatum and brainstem) were dissected on a 0 °C aluminum surface. The specimens were kept frozen at 80 °C. Tissue levels of norepinephrine (NE), dopamine (DA), homovanillic acid (HA), serotonin (5HT) and 5-hydroxyindole acetic acid (5-HIAA) were measured by validated HPLC method. The values summarized in the tables are means ± SD from 8 to 10 animals. 2.2. Determination of the biogenic amines and their metabolites 2.2.1. Chemicals 5-Hydroxytriptamine oxalate salt (5-HT) (Sigma, St. Louis, USA), 5-hydroxy-3-indolacetic acid (5-HIAA) (Sigma) N-methyl-5-hydroxy tryptamine oxalate salt (BST) (Sigma), dopamine hydrochloride (DA) (Sigma), L-norepinephrine-bitartarate (NA) (Serva Heidelberg, Germany), homovanillic acid (HVA) (Sigma) were purchased in the best available quality. Other chemicals were purchased from commercial sources in the best available quality: disodium hydrogen phosphate dihydrate, citric acid monohydrate, ethylenedinitrilotetraacetic acid disodium salt dihydrate
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(Na2EDTA), and acetonitrile (Merck, Darmstadt, Germany), 1-octane sulfonic acid sodium salt (Sigma– Aldrich, Steinheim, Germany), perchloric acid 70% (PCA) (Fluka, Neu-Ulm, Switzerland); phosphoric acid (Sigma). The water was double distilled and deionized and of HPLC grade. 2.3. Sample preparation Samples were treated with four volume of 0.8 M perchloric acid (PCA) homogenized in an Ultra Turrax T25 Janke & Kunkel homogenizer at 20,000 rpm/min for 10 s (IKA Labortechnik, Staufen, Germany), then centrifuged with 14,000g for 10 min at 4 °C in an Eppendorf centrifuge (A. Hettich, Tuttlingen, Germany). The supernatant gained was used for HPLC analysis. Samples were kept at 80 °C before their analysis. Data are given in ng/mg wet tissue ±SD, as indicated. 2.4. Instrumentation Samples were analyzed by reversed phase high-performance liquid chromatography with amperometric/ electrochemical detection (HPLC-EC) consisting from a Jasco pump (PU1580, Tokyo, Japan) connected to a Decade electrochemical detector (Antec, Leyden, Netherlands). Samples were injected directly using a 50 ll loop and separation was carried out at 30 °C, at Eox 0.65 V, with sensitivity of 10 nA on Zorbax RX18 4.6 250 mm (5 lm) analytical column using a Zorbax RX-C18 4.6 12.5 mm (particle size 5 lm), Agilent (USA) pre-column with a time filter of 0.1 s. The mobile phase contained 56.2 mmol/L Na2HPO4, 47.9 mmol/L citric acid, 0.027 mmol/L Na2EDTA, 0.925 mmol/L octane sulfonic acid, and 75:950 acetonitrile/ phosphate buffer. The flow rate of the mobile phase was 1 ml/min. The pH was adjusted to 3.7 with 85% H3PO4. Chromatograms were electronically stored and evaluated using a Borwin 1.21 chromatographic software (JMBS, Le Fontanil, France). 3. Results and discussion In the experiments the progenies of vitamin A or vitamin D imprinted dams were studied. Neither the fertilizing males nor the test-animals were treated. Imprinting of the mothers was performed when newborn and the consequences of this treatment manifested in five brain-areas of the two month’s old F1 generation on the norepinephrine level, on dopaminergic (DA,HVA) and serotonergic (5HT, 5-HIAA) neurotransmission are shown in results. In the brain-stem of F1 generation vitamin A treatment of the mothers resulted in significantly elevated norepinephrine content, but no significant changes in dopaminergic or in serotonergic measures were observed
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K. Tekes et al. / Brain & Development 31 (2009) 666–670
compared to vehicle-treated controls (Table 1). However, after vitamin D treatment significant increase was seen in NE, DA and 5HT tissue levels, while a decrease in 5HIAA. When HVA/DA ratio (as measure of dopamine turnover) was compared to vehicle-treated controls a considerable decrease could be observed (0.5 vs. 1.4, respectively) and that was the case in 5HIAA/5HT ratio (4.8 vs. 10.9). In the hypothalamus imprinting by the vitamins’s caused significant effect only in the serotonergic system by decreasing the 5HIAA tissue levels by both of the vitamins (Table 2). When the 5HIAA/5HT ratios were compared to vehicle-treated controls, it could be seen that vitamin D was less active in decreasing 5HIAA/ 5HT ratio, while vitamin A-caused 5HIAA/5HT ratio decreasing effect was more pronounced if compared to control’s (5.0, 3.4, vs. 8.2, respectively). In the hippocampus only the serotonergic system was touched (Table 3) resulting in significant decrease in 5HT tissue level (by vitamin D) and 5HIAA (by both
vitamins). When 5HIAA/5HT ratios are compared to vehicle-treated controls, in spite of the significant decrease in 5HIAA tissue level after imprinting by vitamin A, considerable increase in serotonergic activity only after vitamin D treatment could be observed (6.6, 6.0, 11.6, respectively). In the striatum only vitamin A caused changes, significantly increasing both DA and 5HIAA levels (Table 4). Vitamin D did not influenced the measured biogenic amine values. HVA/DA ratio was considerably lower in vitamin A – imprinted group compared to controls (0.17 vs. 0.36.). However, in spite of the significant increase in 5HIAA tissue level, increase in 5HIAA/5HT ratio is very similar to control (4.1 vs. 3.2, respectively). The frontal cortex’s dopaminergic and serotonergic system also responded to the imprinting (Table 5). Vitamin A treatment resulted in significant decrease in HVA and 5HIAA tissue levels, however vitamin D imprinting caused decrease in HVA and in 5HT levels. In spite of the unidirectional change in HVA tissue level, the
Table 1 Biogenic amine and metabolite content of the brainstem in adult female rats transgenerationally imprinted by vitamin A or vitamin D (ng/mg wet tissue ± SD). Material
Control
Vitamin A
Significance to control (p)
Vitamin D
Significance to control (p)
NE DA HVA 5HT 5HIAA
301.8 ± 86.4 50.9 ± 10.4 69.6 ± 10.1 111.9 ± 17.4 1220.3 ± 196
417.5 ± 65.3 65.4 ± 46.4 80.99 ± 12.7 127.3 ± 55.4 1037.2 ± 205.6
0.008 n.s. n.s. n.s. n.s.
520.8 ± 113 97.1 ± 46 n.s. 205.7 ± 85.4 992.9 ± 175
0.0003 0.01 n.s. 0.007 0.01
NE, norepinephrine; DA, dopamine; HVA, homovanillic acid; 5HT, serotonin; 5HIAA, 5-hydoxyindoleacetic acid; n.s., not significant compared to vehicle-treated control.
Table 2 Biogenic amine and metabolite content of the hypothalamus in adult female rats transgenerationally imprinted by vitamin A or vitamin D (ng/mg wet tissue ± SD). Material
Control
Vitamin A
Significance to control (p)
Vitamin D
Significance to control (p)
NE DA HVA 5HT 5HIAA
1237.7 ± 303 152.1 ± 42.7 50.8 ± 12.1 91.7 ± 18.9 748.1 ± 262
1154.5 ± 180 183.2 ± 45.2 44.6 ± 5.2 100.8 ± 22.2 342 ± 72.9
n.s. n.s. n.s. n.s. 0.001
1164.6 ± 93 156.6 ± 49.7 46.7 ± 4.9 91.5 ± 33.8 455.5 ± 132
n.s. n.s. n.s. n.s. 0.007
NE, norepinephrine; DA, dopamine; HVA, homovanillic acid; 5HT, serotonin; 5HIAA, 5-hydroxyindoleacetic acid; n.s., not significant compared to vehicle-treated control.
Table 3 Biogenic amine and metabolite content of the hippocampus in adult female rats transgenerationally imprinted by vitamin A or vitamin D (ng/mg wet tissue ± SD). Material
Control
Vitamin A
Significance to control (p)
Vitamin D
Significance to control (p)
DA HVA 5HT 5HIAA
36.5 ± 10.5 47.5 ± 14.9 63.6 ± 17.6 420 ± 25.4
39.4 ± 1.8 73.7 ± 45.2 47.3 ± 14.8 282.6 ± 45.9
n.s. n.s. n.s. 0.00003
31.5 ± 3.1 49.3 ± 3.9 31.2 ± 4.9 361.3 ± 58.5
n.s. n.s. 0.008 0.004
NE, norepinephrine; DA, dopamine; HVA, homovanillic acid; 5HT, serotonin; 5HIAA, 5-hydroxyindoleacetic acid; n.s., not significant compared to vehicle-treated control.
K. Tekes et al. / Brain & Development 31 (2009) 666–670
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Table 4 Biogenic amine and metabolite content of the striatum in adult female rats transgenerationally imprinted by vitamin A or vitamin D (ng/mg wet tissue ± SD). Material
Control
Vitamin A
Significance to control (p)
Vitamin D
Significance to control (p)
DA HVA 5HT 5HIAA
2921.5 ± 983 1067 ± 170.5 110.4 ± 6.07 348.8 ± 76.4
5206.6 ± 303 928 ± 124.4 116.9 ± 15 473.5 ± 69.7
0.0001 n.s. n.s. 0.001
2942 ± 786.7 988.3 ± 77.5 97.9 ± 9.3 421.8 ± 135.4
n.s. n.s. n.s. n.s.
NE, norepinephrine; DA, dopamine; HVA, homovanillic acid; 5HT, serotonin; 5HIAA, 5-hydroxyindoleacetic acid; n.s., not significant compared to vehicle-treated control.
Table 5 Biogenic amine and metabolite content of the frontal cortex in adult female rats transgenerationally imprinted by vitamin A or vitamin D (ng/mg wet tissue ± SD). Material
Control
Vitamin A
Significance to control (p)
Vitamin D
Significance to control (p)
DA HVA 5HT 5HIAA
37.2 ± 7 140.9 ± 22.4 112.5 ± 13.5 538 ± 66.2
46.1 ± 12.8 82.8 ± 19.4 140.8 ± 42.4 433.1 ± 62.6
n.s. 0.001 n.s. 0.002
29.5 ± 3.6 75.7 ± 17.7 42.8 ± 21.8 540 ± 87.2
n.s. 0.0009 0.0000003 n.s.
NE, norepinephrine; DA, dopamine; HVA, homovanillic acid; 5HT, serotonin; 5HIAA, 5-hydroxyindoleacetic acid; n.s., not significant compared to vehicle-treated control.
HVA/DA ratio in vitamin A-treated group is more pronounced than in vitamin D-treated ones compared to controls (1.7 and 2.6, respectively vs. the control 3.8). It is worthwhile to mention that vitamin A imprinting resulted in significant decrease in HIAA, but the 5HIAA/5HT ratio was less sensitive than in vitamin D-treated group if compared to vehicle-treated controls (3.1, 12.6 vs. 4.8). The results show that the effect of neonatal hormonal imprinting on mothers both by vitamin A and vitamin D, the hormonally active vitamins, can be transmitted transgenerationally. Transgenerational consequences of imprinting were also found in many earlier studies, when one of the strongest imprinters, benzpyrene [18], or endorphin or opiates etc. were studied [19,20,23,24]. Recently, many human observations on the transgenerational hormonal imprinting effects were published, e.g. grandparents’ overfeeding influenced grandchildrens’ metabolic syndrome, grandparents’ starvation provoked underweight of grandchildren, and grandmaternal smoking provoked asthma in the F2 generation [25–30]. In an earlier experiment, when the P generation was studied, vitamin A consistently influenced the serotonergic system (5HIAA), while vitamin D the dopaminerg system (DA or HVA) [22]. Vitamin A always reduced the values, vitamin D always increased them. No exception could be seen. In the present case such unidirectional effect cannot be declared. However, the number of significant differences was so high, that the role of chance can be excluded. It must be considered that only one of the parents was imprinted and this could weaken the epigenetic expressivity of hormonal imprinting. Nevertheless, the strength of imprinting is demonstrated and shows that the progenies also react to the perinatal treat-
ment of the mother. In a similar situation, when the effect of vitamin A and D imprinting of the P mothergeneration was studied on the hormone content of F1 generation’s immune cells, the effect was also different in the two generations [31]. However, in this case the change in hormone content was more expressed in the progeny generation than in the mothers’ generation, e.g. histamine level was the third (related to control) in the lymphocytes of vitamin D treated mothers’ offspring, while imprinting with this vitamin was absolutely ineffective in the P generation. This means that not the specific effect is inherited, but the disturbance caused by the hormonal imprinting, i.e. the loosening of the system. The exact mechanism of hormonal imprinting and transgenerational imprinting is still not known. However, the change of methylation pattern of DNA and its epigenetic inheritance is suspected [32–36]. It is important that hormonal imprinting cannot be confounded with other imprinting phenomena (behavioral imprinting, genomic imprinting), although each happens in the perinatal period and in the case of genomic imprinting also methylation processes have a role [37]. Projecting the results to man and considering that the mothers’ or infants’ preventive treatment with receptorlevel acting vitamins is very frequent, even forced, the clinical consequences must be taken into account and the subject is worth further studies. Acknowledgements The authors thank Ms. Katy Kallay and Gyo¨rgyi Guth for their expert technical assistance. Support of Ministry of Health (ETT 343/2006) is highly appreciated.
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Original article
Transgenerational hormonal imprinting caused by vitamin A and vitamin D treatment of newborn rats. Alterations in the biogenic amine contents of the adult brain Korne´lia Tekes a, Melinda Gyenge a, Mo´nika Hantos b, Gyo¨rgy Csaba c,* a Department of Pharmacodynamics, Semmelweis University, H-1445 Budapest, Hungary Department of Pharmacy Administration, Semmelweis University, H-1445 Budapest, Hungary c Department of Genetics, Cell and Immunobiology, Semmelweis University, P.O. Box 370, H-1445 Budapest, Hungary b
Received 16 June 2008; received in revised form 22 October 2008; accepted 23 October 2008
Abstract Biogenic amines (norepinephrine, dopamine, homovanillic acid, serotonin and 5-hyroxyindole acetic acid) were measured by HPLC method in adult F1 generation rats’ brain regions (brainstem, hypothalamus, hippocampus, striatum and frontal cortex), whose mothers (P generation) were treated with vitamin A or vitamin D neonatally (hormonal imprinting). Many significant differences were found, related to the maternally untreated controls. In the earlier studied P generation females, vitamin A consistently influenced the serotonerg system (5HIAA), while vitamin D the dopaminerg system (DA or HVA). Vitamin A imprinting always resulted in reduced, while that by vitamin D always in increased tissue levels. In the present case (directly untreated F1 generation) the transgenerational effect was not unidirectional, however biogenic amine tissue levels were strongly disturbed and brain-area dependent. The results call attention to the transgenerational effect of hormonal imprinting in the case of receptor level acting vitamins which are frequently used in the most imprinting-sensitive period (perinatally) of human life and suggests that caution is warranted. Ó 2008 Elsevier B.V. All rights reserved. Keywords: Neonatal treatment; Transgenerational effect; Vitamins; Biogenic amines; Imprinting
1. Introduction Hormonal imprinting is a well-established phenomenon, that takes place in the developing cells when a hormone meets its target receptor. Usually, it occurs in the perinatal period, but it can happen later as well, when the target cell is in a developing phase [1–3]. As a result of imprinting the binding capacity of the receptor is modulated reaching its value characteristic for the adult animal [4]. Without imprinting there is no normal receptor maturation [5] however, the over-abundance of the target hormone or even foreign molecules that are able *
Corresponding author. Tel./fax: +36 1 210 2950. E-mail address: [email protected] (G. Csaba).
0387-7604/$ - see front matter Ó 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.braindev.2008.10.007
to bind to the receptor, can cause ‘‘faulty imprinting” [1–4] with life-long genetic, morphological, biochemical (alteration of cell proliferation, change in hormone production) and behavioral (loss of libido, aggressivity) consequences [6–14]. The effect of imprinting is not limited on the direct targets but – by a presently unknown mechanism – it can also have effect on other members of the endocrine system [15–17]. The consequences of imprinting can be transmitted to the progeny generations as well, and even in the F1 and F2 could be observed [18–20]. The behavioral effects of imprinting called our attention to the possibility of the influence of imprinting to brain targets, which has been suggested by many previous experiments [21].
K. Tekes et al. / Brain & Development 31 (2009) 666–670
Previously, we studied the imprinting effect of vitamins A and D, two molecules that act by binding to receptors belonging to the steroid receptor superfamily. Both compounds are used widely during the perinatal critical period as preventive treatments. The effect of these compounds on the biogenic amine neurotransmitter levels in the brain was demonstrated [22]. Considering these results, the present experiment was addressed to the transgenerational imprinting effect by these vitamins on the biogenic amine tissue levels in the brain. 2. Materials and methods 2.1. Animals and treatment Female newborn (strictly before the age of 24 h) Charles River originated Wistar rats (P generation) of our closed breeding colony were treated with a single dose of 0.05 mg vitamin D3 (cholecalciferol, Sigma, USA) or with 3 mg vitamin A (retinol, EGIS Budapest, Hungary) solved in 0.1 ml sunflower seed oil. Compounds were given subcutaneously under the skin of the neck. Controls got the vehicle. The animals were housed at room temperature under normal light cycle. Food and water were available ad libidum. When the animals were 3 months old they were mated with control (untreated) males. Two months old female progenies (F1 generation) were used for the experiments. All of the animal experiments were executed according to the Council Directive No. 86/609/EEC. Animals under ether anesthesia were sacrificed by decapitation, brains immediately removed and five regions (frontal cortex, hypothalamus, hippocampus, striatum and brainstem) were dissected on a 0 °C aluminum surface. The specimens were kept frozen at 80 °C. Tissue levels of norepinephrine (NE), dopamine (DA), homovanillic acid (HA), serotonin (5HT) and 5-hydroxyindole acetic acid (5-HIAA) were measured by validated HPLC method. The values summarized in the tables are means ± SD from 8 to 10 animals. 2.2. Determination of the biogenic amines and their metabolites 2.2.1. Chemicals 5-Hydroxytriptamine oxalate salt (5-HT) (Sigma, St. Louis, USA), 5-hydroxy-3-indolacetic acid (5-HIAA) (Sigma) N-methyl-5-hydroxy tryptamine oxalate salt (BST) (Sigma), dopamine hydrochloride (DA) (Sigma), L-norepinephrine-bitartarate (NA) (Serva Heidelberg, Germany), homovanillic acid (HVA) (Sigma) were purchased in the best available quality. Other chemicals were purchased from commercial sources in the best available quality: disodium hydrogen phosphate dihydrate, citric acid monohydrate, ethylenedinitrilotetraacetic acid disodium salt dihydrate
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(Na2EDTA), and acetonitrile (Merck, Darmstadt, Germany), 1-octane sulfonic acid sodium salt (Sigma– Aldrich, Steinheim, Germany), perchloric acid 70% (PCA) (Fluka, Neu-Ulm, Switzerland); phosphoric acid (Sigma). The water was double distilled and deionized and of HPLC grade. 2.3. Sample preparation Samples were treated with four volume of 0.8 M perchloric acid (PCA) homogenized in an Ultra Turrax T25 Janke & Kunkel homogenizer at 20,000 rpm/min for 10 s (IKA Labortechnik, Staufen, Germany), then centrifuged with 14,000g for 10 min at 4 °C in an Eppendorf centrifuge (A. Hettich, Tuttlingen, Germany). The supernatant gained was used for HPLC analysis. Samples were kept at 80 °C before their analysis. Data are given in ng/mg wet tissue ±SD, as indicated. 2.4. Instrumentation Samples were analyzed by reversed phase high-performance liquid chromatography with amperometric/ electrochemical detection (HPLC-EC) consisting from a Jasco pump (PU1580, Tokyo, Japan) connected to a Decade electrochemical detector (Antec, Leyden, Netherlands). Samples were injected directly using a 50 ll loop and separation was carried out at 30 °C, at Eox 0.65 V, with sensitivity of 10 nA on Zorbax RX18 4.6 250 mm (5 lm) analytical column using a Zorbax RX-C18 4.6 12.5 mm (particle size 5 lm), Agilent (USA) pre-column with a time filter of 0.1 s. The mobile phase contained 56.2 mmol/L Na2HPO4, 47.9 mmol/L citric acid, 0.027 mmol/L Na2EDTA, 0.925 mmol/L octane sulfonic acid, and 75:950 acetonitrile/ phosphate buffer. The flow rate of the mobile phase was 1 ml/min. The pH was adjusted to 3.7 with 85% H3PO4. Chromatograms were electronically stored and evaluated using a Borwin 1.21 chromatographic software (JMBS, Le Fontanil, France). 3. Results and discussion In the experiments the progenies of vitamin A or vitamin D imprinted dams were studied. Neither the fertilizing males nor the test-animals were treated. Imprinting of the mothers was performed when newborn and the consequences of this treatment manifested in five brain-areas of the two month’s old F1 generation on the norepinephrine level, on dopaminergic (DA,HVA) and serotonergic (5HT, 5-HIAA) neurotransmission are shown in results. In the brain-stem of F1 generation vitamin A treatment of the mothers resulted in significantly elevated norepinephrine content, but no significant changes in dopaminergic or in serotonergic measures were observed
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compared to vehicle-treated controls (Table 1). However, after vitamin D treatment significant increase was seen in NE, DA and 5HT tissue levels, while a decrease in 5HIAA. When HVA/DA ratio (as measure of dopamine turnover) was compared to vehicle-treated controls a considerable decrease could be observed (0.5 vs. 1.4, respectively) and that was the case in 5HIAA/5HT ratio (4.8 vs. 10.9). In the hypothalamus imprinting by the vitamins’s caused significant effect only in the serotonergic system by decreasing the 5HIAA tissue levels by both of the vitamins (Table 2). When the 5HIAA/5HT ratios were compared to vehicle-treated controls, it could be seen that vitamin D was less active in decreasing 5HIAA/ 5HT ratio, while vitamin A-caused 5HIAA/5HT ratio decreasing effect was more pronounced if compared to control’s (5.0, 3.4, vs. 8.2, respectively). In the hippocampus only the serotonergic system was touched (Table 3) resulting in significant decrease in 5HT tissue level (by vitamin D) and 5HIAA (by both
vitamins). When 5HIAA/5HT ratios are compared to vehicle-treated controls, in spite of the significant decrease in 5HIAA tissue level after imprinting by vitamin A, considerable increase in serotonergic activity only after vitamin D treatment could be observed (6.6, 6.0, 11.6, respectively). In the striatum only vitamin A caused changes, significantly increasing both DA and 5HIAA levels (Table 4). Vitamin D did not influenced the measured biogenic amine values. HVA/DA ratio was considerably lower in vitamin A – imprinted group compared to controls (0.17 vs. 0.36.). However, in spite of the significant increase in 5HIAA tissue level, increase in 5HIAA/5HT ratio is very similar to control (4.1 vs. 3.2, respectively). The frontal cortex’s dopaminergic and serotonergic system also responded to the imprinting (Table 5). Vitamin A treatment resulted in significant decrease in HVA and 5HIAA tissue levels, however vitamin D imprinting caused decrease in HVA and in 5HT levels. In spite of the unidirectional change in HVA tissue level, the
Table 1 Biogenic amine and metabolite content of the brainstem in adult female rats transgenerationally imprinted by vitamin A or vitamin D (ng/mg wet tissue ± SD). Material
Control
Vitamin A
Significance to control (p)
Vitamin D
Significance to control (p)
NE DA HVA 5HT 5HIAA
301.8 ± 86.4 50.9 ± 10.4 69.6 ± 10.1 111.9 ± 17.4 1220.3 ± 196
417.5 ± 65.3 65.4 ± 46.4 80.99 ± 12.7 127.3 ± 55.4 1037.2 ± 205.6
0.008 n.s. n.s. n.s. n.s.
520.8 ± 113 97.1 ± 46 n.s. 205.7 ± 85.4 992.9 ± 175
0.0003 0.01 n.s. 0.007 0.01
NE, norepinephrine; DA, dopamine; HVA, homovanillic acid; 5HT, serotonin; 5HIAA, 5-hydoxyindoleacetic acid; n.s., not significant compared to vehicle-treated control.
Table 2 Biogenic amine and metabolite content of the hypothalamus in adult female rats transgenerationally imprinted by vitamin A or vitamin D (ng/mg wet tissue ± SD). Material
Control
Vitamin A
Significance to control (p)
Vitamin D
Significance to control (p)
NE DA HVA 5HT 5HIAA
1237.7 ± 303 152.1 ± 42.7 50.8 ± 12.1 91.7 ± 18.9 748.1 ± 262
1154.5 ± 180 183.2 ± 45.2 44.6 ± 5.2 100.8 ± 22.2 342 ± 72.9
n.s. n.s. n.s. n.s. 0.001
1164.6 ± 93 156.6 ± 49.7 46.7 ± 4.9 91.5 ± 33.8 455.5 ± 132
n.s. n.s. n.s. n.s. 0.007
NE, norepinephrine; DA, dopamine; HVA, homovanillic acid; 5HT, serotonin; 5HIAA, 5-hydroxyindoleacetic acid; n.s., not significant compared to vehicle-treated control.
Table 3 Biogenic amine and metabolite content of the hippocampus in adult female rats transgenerationally imprinted by vitamin A or vitamin D (ng/mg wet tissue ± SD). Material
Control
Vitamin A
Significance to control (p)
Vitamin D
Significance to control (p)
DA HVA 5HT 5HIAA
36.5 ± 10.5 47.5 ± 14.9 63.6 ± 17.6 420 ± 25.4
39.4 ± 1.8 73.7 ± 45.2 47.3 ± 14.8 282.6 ± 45.9
n.s. n.s. n.s. 0.00003
31.5 ± 3.1 49.3 ± 3.9 31.2 ± 4.9 361.3 ± 58.5
n.s. n.s. 0.008 0.004
NE, norepinephrine; DA, dopamine; HVA, homovanillic acid; 5HT, serotonin; 5HIAA, 5-hydroxyindoleacetic acid; n.s., not significant compared to vehicle-treated control.
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Table 4 Biogenic amine and metabolite content of the striatum in adult female rats transgenerationally imprinted by vitamin A or vitamin D (ng/mg wet tissue ± SD). Material
Control
Vitamin A
Significance to control (p)
Vitamin D
Significance to control (p)
DA HVA 5HT 5HIAA
2921.5 ± 983 1067 ± 170.5 110.4 ± 6.07 348.8 ± 76.4
5206.6 ± 303 928 ± 124.4 116.9 ± 15 473.5 ± 69.7
0.0001 n.s. n.s. 0.001
2942 ± 786.7 988.3 ± 77.5 97.9 ± 9.3 421.8 ± 135.4
n.s. n.s. n.s. n.s.
NE, norepinephrine; DA, dopamine; HVA, homovanillic acid; 5HT, serotonin; 5HIAA, 5-hydroxyindoleacetic acid; n.s., not significant compared to vehicle-treated control.
Table 5 Biogenic amine and metabolite content of the frontal cortex in adult female rats transgenerationally imprinted by vitamin A or vitamin D (ng/mg wet tissue ± SD). Material
Control
Vitamin A
Significance to control (p)
Vitamin D
Significance to control (p)
DA HVA 5HT 5HIAA
37.2 ± 7 140.9 ± 22.4 112.5 ± 13.5 538 ± 66.2
46.1 ± 12.8 82.8 ± 19.4 140.8 ± 42.4 433.1 ± 62.6
n.s. 0.001 n.s. 0.002
29.5 ± 3.6 75.7 ± 17.7 42.8 ± 21.8 540 ± 87.2
n.s. 0.0009 0.0000003 n.s.
NE, norepinephrine; DA, dopamine; HVA, homovanillic acid; 5HT, serotonin; 5HIAA, 5-hydroxyindoleacetic acid; n.s., not significant compared to vehicle-treated control.
HVA/DA ratio in vitamin A-treated group is more pronounced than in vitamin D-treated ones compared to controls (1.7 and 2.6, respectively vs. the control 3.8). It is worthwhile to mention that vitamin A imprinting resulted in significant decrease in HIAA, but the 5HIAA/5HT ratio was less sensitive than in vitamin D-treated group if compared to vehicle-treated controls (3.1, 12.6 vs. 4.8). The results show that the effect of neonatal hormonal imprinting on mothers both by vitamin A and vitamin D, the hormonally active vitamins, can be transmitted transgenerationally. Transgenerational consequences of imprinting were also found in many earlier studies, when one of the strongest imprinters, benzpyrene [18], or endorphin or opiates etc. were studied [19,20,23,24]. Recently, many human observations on the transgenerational hormonal imprinting effects were published, e.g. grandparents’ overfeeding influenced grandchildrens’ metabolic syndrome, grandparents’ starvation provoked underweight of grandchildren, and grandmaternal smoking provoked asthma in the F2 generation [25–30]. In an earlier experiment, when the P generation was studied, vitamin A consistently influenced the serotonergic system (5HIAA), while vitamin D the dopaminerg system (DA or HVA) [22]. Vitamin A always reduced the values, vitamin D always increased them. No exception could be seen. In the present case such unidirectional effect cannot be declared. However, the number of significant differences was so high, that the role of chance can be excluded. It must be considered that only one of the parents was imprinted and this could weaken the epigenetic expressivity of hormonal imprinting. Nevertheless, the strength of imprinting is demonstrated and shows that the progenies also react to the perinatal treat-
ment of the mother. In a similar situation, when the effect of vitamin A and D imprinting of the P mothergeneration was studied on the hormone content of F1 generation’s immune cells, the effect was also different in the two generations [31]. However, in this case the change in hormone content was more expressed in the progeny generation than in the mothers’ generation, e.g. histamine level was the third (related to control) in the lymphocytes of vitamin D treated mothers’ offspring, while imprinting with this vitamin was absolutely ineffective in the P generation. This means that not the specific effect is inherited, but the disturbance caused by the hormonal imprinting, i.e. the loosening of the system. The exact mechanism of hormonal imprinting and transgenerational imprinting is still not known. However, the change of methylation pattern of DNA and its epigenetic inheritance is suspected [32–36]. It is important that hormonal imprinting cannot be confounded with other imprinting phenomena (behavioral imprinting, genomic imprinting), although each happens in the perinatal period and in the case of genomic imprinting also methylation processes have a role [37]. Projecting the results to man and considering that the mothers’ or infants’ preventive treatment with receptorlevel acting vitamins is very frequent, even forced, the clinical consequences must be taken into account and the subject is worth further studies. Acknowledgements The authors thank Ms. Katy Kallay and Gyo¨rgyi Guth for their expert technical assistance. Support of Ministry of Health (ETT 343/2006) is highly appreciated.
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