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Melamine in prenatal and postnatal organs in rats
Reproductive Toxicology 35 (2013) 40–47
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Reproductive Toxicology journal homepage: www.elsevier.com/locate/reprotox
Melamine in prenatal and postnatal organs in rats Ching Yan Chu a , Kai On Chu a,b , Chung Shun Ho c , Sung Shing Kwok c , Ho Ming Chan c , Kwok Pui Fung d , Chi Chiu Wang a,d,∗ a
Department of Obstetrics and Gynaecology, Hong Kong Department of Ophthalmology and Visual Sciences, The Chinese University of Hong Kong, Hong Kong Department of Chemical Pathology, Prince of Wales Hospital, Hong Kong d School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong b c
a r t i c l e
i n f o
Article history: Received 24 February 2012 Received in revised form 5 June 2012 Accepted 15 June 2012 Available online 23 June 2012 Keywords: Melamine Pharmacokinetics Distribution Pregnancy Infants
a b s t r a c t Melamine can be transferred to fetus in utero through placenta and to infant ex utero by breast feeding. In this study, we characterized the pharmacokinetics of melamine in prenatal and postnatal organs in rats. Single bolus of melamine was administered to pregnant rats at different gestational stages and to infants at different postnatal stages. Distribution of melamine in maternal serum was about 30% higher in late pregnancy than that in early pregnancy; and it was 2 folds higher in postnatal serum in early infants in young adulthood. Distribution of melamine in all postnatal organs was higher than that in prenatal organs. Postnatal kidneys in early infants had the highest maximum concentration and the lowest clearance of melamine than the other postnatal organs. It may relate to the high vulnerability to the toxicity of melamine in this population. © 2012 Elsevier Inc. All rights reserved.
1. Introduction Nephrotoxic melamine (2,4,6-triamino-1,3,5-triazine) has been contaminating food for years. It was introduced to food sources as one of the adulteration with high nitrogen content in dairy products. The intention was to deceive the routine tests like Kjeldahl reaction [1] that cannot distinguish nitrogen atoms of a proteinaceous source from the others, such as amides, ammonium, alkaloids, urea, as well as melamine. The deliberate adulteration of pet food and infant formula by melamine, resulting in death of pets
Abbreviations: ACN, acetonitrile; AUC, area under the curve; AUMC, predicted area under the first moment curve divided by the predicted AUC; Cl/F, apparent oral clearance; Cmax , maximum concentration; FDA, The US Food and Drug Administration; GFR, glomerular filtration rate; Kin , absorption rate constant; Kout , elimination rate constant; z , terminal elimination rate; LC–MS, liquid chromatography–mass spectrometry; MRTinf , mean residence time extrapolated to infinity; T1/2 , half-life; Tmax , time of maximum concentration; Vz /F, apparent volume of distribution. ∗ Corresponding author at: Department of Obstetrics and Gynaecology, The Chinese University of Hong Kong, Shatin, Hong Kong. Tel.: +852 2632 2810; fax: +852 2636 0008. E-mail addresses: nina [email protected] (C.Y. Chu), [email protected] (K.O. Chu), [email protected] (C.S. Ho), [email protected] (S.S. Kwok), [email protected] (H.M. Chan), [email protected] (K.P. Fung), [email protected] (C.C. Wang). 0890-6238/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.reprotox.2012.06.003
and acute renal failure in human, has raised immense concern on the safety issue worldwide [2–4]. Melamine is not metabolized in human body. It is excreted in urine per se [5,6]. It has low direct toxicity to human but its low solubility is believed to be the culprit of its damage to kidneys. When melamine forms a complex with cyanuric acid, which is almost undissolvable in water, it easily blocks the renal tubules [2]. A communiqué in 2008 stated that tens of thousands of children were hospitalized after taking in the adulterated baby formula in the People Republic of China. Most of the affected children have been diagnosed with anuria, kidney stones, urinary tract stones, hydronephrosis, even more severe cases with acute renal failure and death were reported [3]. Besides kidney, melamine was also detected in other tissues, e.g., liver, spleen, bladder and brain [7]. Developing organs in fetus in utero and neonates ex utero are highly susceptible to the melamine accumulation [8]. In this study, we would like to investigate the detailed pharmacokinetics of melamine in prenatal and postnatal organs at different developmental stages. 2. Method 2.1. Chemicals Melamine for animal experiments and solvents for LC–MS analytical measurement were purchased from Sigma Chemical Co (St. Louis, MO). 13 C3 ,15 N3 -melamine (Cambridge Isotope Laboratories, Andover, MA) was used as the internal standard.
C.Y. Chu et al. / Reproductive Toxicology 35 (2013) 40–47 2.2. Animals In order to determine the pharmacokinetic of melamine in vivo, pregnant and postnatal Sprague-Dawley rats were used. Animals were obtained from the Laboratory Animal Services Center of the Chinese University of Hong Kong. Ethics approval for the use and care of animal study was obtained from the Animal Ethics Committee of the University. All animals were housed at 25 ◦ C with a 12-h light/dark cycle with free access of standard water and chow. Young pups were housed with lactating rats before weaning.
2.3. Dosage Both the prenatal and the postnatal rats were investigated. All animals, except lactating rats, received a single oral bolus of melamine at 21.4 mg/kg of body weight as previously described [8]. The dosage for rat was chosen as 5.5 fold of the equivalent dose of human tolerable daily intake (TDI) of melamine recommended by the US Food and Drug Administration (FDA) in 2008 [9] and converted by the online FDA Dose Calculator [10]. The dosage conversion from human dose to rat dose included height, weight and surface area and was corrected by conversion factor, i.e. 6, as previously described [11]. The selected dose is also equivalent to the melamine levels detected in the contaminated dairy food products in the local area [12].
2.4. Administration Pregnant rats at the gestational day 10, 15 and 20 (E10, E15 and E20) and infant rats at postnatal week 2, 4, 6 and 8 (P2W, P4W, P6W and P8W) were weight and gavaged with melamine in distilled water. Lactating mothers were not administered the melamine during the experiments for postnatal pups. All animals were sacrificed and samples were collected at 0, 0.5, 1, 2, 3, 4, 5, 6, 7, 8 and 24 h after administration. Maternal and infant serum were prepared from peripheral whole blood from inferior vena cavae at all gestational and postnatal periods, except decapitation for the 2-week old neonates. Serum were centrifuged at 3000 rpm, at 4 ◦ C for 10 min. Fetal kidneys at E15 and, fetal kidneys, livers, lungs, hearts and brains at E20 were collected. Each of the prenatal organs of the same litter was pooled together as a single sample. Postnatal kidneys were collected in all the postnatal periods, while postnatal livers, lungs, hearts, spleens and brains were also collected in 2-week old rats. All tissues were washed in PBS and all samples were frozen at −80 ◦ C prior to preparation for melamine quantitation by LC–MS. N = 5 for each time point, i.e. total sample number of animals for each developmental stage were 55.
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The half-lives (T1/2 ) of melamine in various samples were estimated by the z and the Kout , the product of ln 2 and the reciprocal of z or Kout for experimental comparison. T1/2 =
ln 2 z
or
T1/2 =
ln 2 z
2.7. Statistical analysis Statistical analysis of Tmax , Cmax , z , T1/2 , MRTinf , Cl/F and Vz /F of the maternal serum of the pregnant rats versus that of the non-pregnant adult control and the infant serum at other stages versus that at P2W were carried out by one-way ANOVA. T-tests assuming equal variants (2-tailed) were followed when there was significance detected by ANOVA. One-way ANOVA was also applied to organs of the same prenatal and postnatal stages on Tmax , Cmax , and AUC. Pharmacokinetic parameters between the prenatal and the postnatal kidneys were compared by T-test.
3. Results 3.1. Maternal and postnatal serum All maternal and postnatal serum followed the noncompartmental pharmacokinetic model. Single peak was obtained from the melamine distribution over the 24-h period. The pharmacokinetic parameters were shown in Table 1. The pharmacokinetic profiles in maternal serum were similar in non-pregnant and pregnant rat at E20, but different from those of E10 and E15. The pharmacokinetic profiles of postnatal serum at P4W, P6W and P8W were similar, but not to that of P2W rats (Fig. 1). An
2.5. Quantitation of melamine Acetonitrile (ACN)/water (50:50, v/v) was used for tissue homogenization and melamine extraction from tissues. Sera required no extraction. Melamine in both tissue homogenates and sera were analyzed by UPLC-MS Waters Xevo TQ System (Waters, Milford, MA). Extraction and measurement of melamine were performed as described previously [8]. The limit of quantitation was 5 ppb and the linearity was up to 10,000 ppb. The recovery of the assay was 105 ± 13%.
2.6. Pharmacokinetic analysis Pharmacokinetic analysis was carried out by WINNONLIN Professional Version 4.01 (Pharsight Corp, MO). Maximum concentration (Cmax ), time of Cmax (Tmax ) and area under the curve (AUC) of melamine in each sample was computed. Exposure of melamine was presented as normalized AUC calculated by the trapezoidal formula in terms of ng/g h. The average time of melamine staying in the samples from zero time point to infinity (mean residence time extrapolated to infinity, MRTinf ) was obtained from the predicted area under the first moment curve divided by the predicted AUC (AUMC) from zero time point to infinity. MRTinf =
AUMCinf AUCinf
The overall elimination rate of the melamine calculated from the terminal slope was expressed as terminal elimination rate (z ) which described the elimination rate of the molecule following non-compartment model. Absorption rate constant (Kin ) as the rate constant for the oral melamine intake and elimination rate (Kout ) as the elimination rate constant of melamine were obtained from first order model. Apparent oral clearance (Cl/F) and apparent volume of distribution (Vz /F) were determined from the terminal slope. Cl/F =
Dose AUCinf
Vz /F =
Dose z × AUCinf
Fig. 1. Distribution of melamine in maternal and postnatal sera. Maternal serum samples (upper panel) from non-pregnant and pregnant rats at gestational day 10 (E10), day 15 (E15) and day 20 (E20), N = 5 per time point and postnatal serum samples (lower panel) from postnatal rats at 2 weeks (P2W), 4 weeks (P4W), 6 weeks (P6W) and 8 weeks (P8W), N = 5 per time point, are shown. Mean ± SD are presented.
n = 5 per time point, total N = 55 per group. Tmax , maximum peak concentration time of the prominent peak; Cmax , maximum concentration of the prominent peak; AUC, area under the curve integrate normalized to molarity; z , terminal slope or terminal elimination rate; T1/2 , half live (estimated from z ); MRTinf , mean residence time from time zero to infinity; Cl/F, apparent clearance; and Vz /F, apparent volume of distribution. Data shows mean ± SD values. N/A, data not applicable. † Post hoc test: p < 0.05 comparing maternal serum with non-pregnant rats serum. ‡ Post hoc test: p < 0.05 comparing postnatal sera with the P2W serum.
N = 55 N = 55
Non-compartment 0.88 ± 0.25 10,315.75 ± 546.34 32,838.17 ± 1824.92‡ 0.32 ± 0.09‡ 2.29 ± 0.52‡ 2.76 ± 0.17‡ 0.65 ± 0.04 2.16 ± 0.50
N = 55
Non-compartment 0.8 ± 0.27 9911.80 ± 1881.75 26,695.55 ± 1603.62‡ 0.54 ± 0.06‡ 1.29 ± 0.13‡ 2.33 ± 0.21‡ 0.80 ± 0.05 1.51 ± 0.23
N = 55
Non-compartment 1.80 ± 1.89 12,242.80 ± 3112.51 72,177.15 ± 7004.65 0.17 ± 0.05 4.26 ± 1.15 6.53 ± 1.72 0.30 ± 0.03 1.82 ± 0.46
N = 55 N = 55 N = 55
Non-compartment 0.90 ± 0.22 10,609.80 ± 1685.23† 34,860.0 ± 2291.41† 0.38 ± 0.03† 1.85 ± 0.13† 3.0 ± 0.22 0.62 ± 0.04† 1.64 ± 0.13 Non-compartment 0.75 ± 0.29 14,465.25 ± 1380.63 50,975.13 ± 1849.49 0.28 ± 0.03 2.55 ± 0.34 3.32 ± 0.27 0.42 ± 0.02 1.55 ± 0.22 Model Tmax (h) Cmax (g/kg) AUC (h g/kg) z (1/h) T1/2 (h) MRTinf (h) Cl/F (1/h) Vz /F
N = 55
Non-compartment 1.20 ± 1.04 11,581.20 ± 2700.51 39,138.43 ± 2809.09† 0.26 ± 0.02 2.70 ± 0.18 3.34 ± 0.34 0.55 ± 0.04† 2.14 ± 0.23†
Non-compartment 1.0 ± 0.0 14,308.0 ± 1471.76 51,447.58 ± 2160.24 0.32 ± 0.04 2.19 ± 0.24 3.53 ± 0.33 0.42 ± 0.02 1.32 ± 0.16
P6W P4W Postnatal Serum
P2W E20 E15 E10 Non-pregnant
Maternal serum
Table 1 Pharmacokinetic parameters of melamine in maternal and postnatal sera.
Non-compartment 0.88 ± 0.25 11,355.50 ± 3185.49 34,483.97 ± 8664.15‡ 0.35 ± 0.09‡ 2.08 ± 0.52‡ 2.96 ± 0.19‡ 0.65 ± 0.15 1.89 ± 0.38
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P8W
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Fig. 2. Tmax , Cmax and AUC of melamine in maternal and postnatal sera. Maternal serum samples (red bars) from non-pregnant and pregnant rats at gestational day 10 (E10), day 15 (E15) and day 20 (E20), per time point, and postnatal serum samples (blue bars) from postnatal rats at 2 week (P2W), 4 weeks (P4W), 6 weeks (P6W) and 8 weeks (P8W), N = 5 per time point, are shown. Mean are presented. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
increase in Tmax of maternal serum melamine was observed from the non-pregnant to the pregnant rat at E15 (1.6 times of that of that non-pregnant rat), and then dropped at E20 (1.3 times) (Fig. 2). Postnatal serum had the highest Tmax of melamine at P2W, and then decreased by half and maintained at this level for the following 6 weeks (0.44–0.48 times). Comparing the Tmax of serum melamine of P8W and that of the non-pregnant adult rat, there was a further but slight drop observed, with no significant difference. Cmax of serum melamine of non-pregnant rat was the highest (Fig. 2). During the course of pregnancy, Cmax of melamine rose; in late pregnancy, Cmax became comparable to that of the non-pregnant one. Cmax of melamine of maternal serum were 0.73, 0.80 and 0.99 fold at E10, E15 and E20, compared to that of the non-pregnant ones. Cmax of serum melamine of pregnant rat at E10 showed a significant difference to the non-pregnant rat and the non-pregnant rats at E20 (p < 0.01). Cmax of melamine of postnatal serum became the highest at P2W. Cmax of melamine of rats at P4W, P6W and P8W were 0.81, 0.84 and 0.93 fold of that of rat at P2W, respectively. Similar trends were obtained in the AUC of serum melamine of the neonates (0.37–0.48 fold of that of rat at postnatal week 2) (Fig. 2). AUC of serum melamine of postnatal
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followed by prenatal kidney, lung, heart and liver (Fig. 4). The Tmax of melamine of these prenatal organs were 0.73, 0.42, 0.40 and 0.39 times of that of prenatal brain, respectively. Pharmacokinetic analysis of melamine in prenatal liver showed the highest Cmax and AUC (Fig. 4). Cmax of melamine of prenatal organs in descending order were liver, heart (0.82 fold), lung (0.80 fold), kidney (0.75 fold) and brain (0.65 fold of that of liver); AUC of melamine of prenatal organs in descending order were liver, brain (0.85 fold), kidney (0.84 fold), lung (0.83 fold), and heart (0.78 fold of that of liver). Melamine measured in the postnatal kidney at P2W had the highest Tmax , Cmax and AUC among all other postnatal organs collected (Fig. 4). Tmax of melamine in postnatal brain was also high, i.e., 0.8 fold of that of postnatal kidney. On the other hand, peaks of the melamine profile of the other postnatal organs were observed much earlier in postnatal heart (0.35 times), lung (0.27 times), spleen (0.20 times) and liver (0.18 times of kidney), in descending order. Despite of the high Tmax of melamine in postnatal brain, the other 2 parameters, Cmax and AUC of melamine in postnatal brain were comparable to those of the other organs (Fig. 4). Cmax and AUC of melamine in postnatal kidney were particularly high. Cmax of melamine in postnatal lung, liver, spleen, heart and brain of rat at P2W were only 0.22, 0.19, 0.18, 0.15 and 0.10 of that of postnatal kidney, respectively. AUC of melamine had the same descending order of that of Cmax . Those of postnatal lung, liver, spleen, heart and brain were observed to be only 0.34, 0.31, 0.29, 0.29 and 0.22 of that of postnatal kidney. Tmax of melamine in postnatal kidney at P2W (p < 0.05), Cmax of melamine in postnatal liver, heart and brain (p < 0.005) and postnatal kidney (p < 0.01), and AUC of all postnatal organs (p < 0.005) were significantly higher than that in the prenatal organs at E20. 3.3. Prenatal and postnatal kidneys
rats at 2 week was significantly different from that of the other 3 postnatal groups as well as from that of the non-pregnant adult (p < 0.005). Non-pregnant adult also showed a very significantly different AUC of serum melamine from that of the postnatal rats (p < 0.005), except those of P8W (p < 0.01). AUC of serum melamine of the pregnant adults increased gradually throughout the pregnancy. In the later stage of pregnancy, the AUC of serum melamine was similar to that of the non-pregnant adult (0.68–1.01 fold of that of the non-pregnant rat, from E10 to E20). AUC of serum melamine of both of the non-pregnant rats and the pregnant rats at E20 day showed a big significant difference against that of the pregnant rats at earlier stages (p < 0.005).
Since distinct pharmacokinetic profiles of melamine in prenatal and postnatal kidneys were observed, pharmacokinetic properties in the developing kidneys between E20 and P2W were further compared (Fig. 5 and Table 3). Both the Cmax and the AUC of melamine in prenatal kidneys significantly lower than that in postnatal kidneys (p < 0.005). Tmax and AUC of melamine in postnatal kidneys were the highest (Fig. 4). Tmax of melamine of kidneys at P4W, P6W and P8W were detected much earlier, only 0.35, 0.18 and 0.14 times of that of rat at P2W; also AUC of melamine of kidneys at P4W, P6W and P8W only 0.53, 0.44 and 0.38 fold of that of rat at P2W, respectively (Fig. 4). Tmax of melamine in kidneys of postnatal rat at P2W is significantly higher from the other postnatal groups (p < 0.05); AUC of melamine in kidney at P2W is also significantly higher from that at P6W and P8W (p < 0.005). Cmax displayed a different trend, having that of the rats at week 2 and 4 higher than the rest, and that of rats of P6W the lowest (Fig. 4). Cmax of melamine of rats at P4W, P6W and P8W were 0.98, 0.46 and 0.59 fold of that of rat at P2W, respectively.
3.2. Prenatal and postnatal organs
4. Discussion
Distribution of melamine in maternal serum was the highest in E20 and so as that of in the postnatal serum at P2W. Further pharmacokinetic comparison in prenatal and postnatal organs was done between these 2 developmental stages. Pharmacokinetics of melamine of prenatal liver, lung and heart at E20 followed 1compartment model while the other prenatal organs of the same stage, i.e., kidney and brain, as well as those of the postnatal rats at 2 week, followed the non-compartmental model. In each of the profiles, only 1 peak was observed (Fig. 3). Pharmacokinetic parameters are shown in Table 2. Tmax of melamine of prenatal brain of E20 rats was the highest among the 5 prenatal organs we had collected,
During the course of pregnancy, maternal blood volume increases. Glomerular filtration rate (GFR), salt and water reabsorption increase dramatically during the early pregnancy and last until at least mid-pregnancy. Therefore, volume of body fluid increases, most rapidly during the mid-pregnancy. Toward the later stage of pregnancy, GFR decreases slightly while the reabsorption of salt and water continue to be high [13]. As a consequence, more melamine can be excreted by the maternal kidney, especially during the early and the mid-pregnancy. The dilution effect, or change in distribution of maternal melamine, reflected by the pharmacokinetic parameters Cmax and AUC of the pregnant rats in early stages of
Fig. 3. Distribution of melamine in prenatal and postnatal organs. Prenatal organs (upper panel), N = 5 per time point, from gestational day 20 and postnatal organs (lower panel), N = 5 per time point, from postnatal week 2 are shown. Mean ± SD are presented.
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Table 2 Pharmacokinetic parameters of melamine in prenatal organs and postnatal organs. Fetal organs at E20
Model Tmax (h) Cmax (g/kg) AUC (h g/kg) z (1/h) Kin (1/h) Kout (1/h) T1/2 (h) MRTinf (h) Cl/F (1/h) Vz /F
Kidney N = 55
Liver N = 55
Lung N = 55
Heart N = 55
Brain N = 55
Non-compartment 2.20 ± 0.45 548.83 ± 70.32 3029.02 ± 441.47 0.23 ± 0.06 N/A N/A 3.19 ± 0.92 5.15 ± 1.10 7.17 ± 0.93 32.32 ± 7.01
1-Compartment 1.18 ± 0.26† 735.70 ± 86.46† 3611.24 ± 420.28 N/A 3.95 ± 2.25 0.26 ± 0.05 2.74 ± 0.42 N/A 5.98 ± 0.64 23.68 ± 4.64
1-Compartment 1.25 ± 0.22† 589.12 ± 50.61 2980.0 ± 159.24 N/A 3.09 ± 1.31 0.25 ± 0.03 2.77 ± 0.31 N/A 7.20 ± 0.37 28.87 ± 4.17
1-Compartment 1.21 ± 0.34† 600.87 ± 17.77 2833.29 ± 147.95 N/A 2.82 ± 1.44 0.28 ± 0.03 2.47 ± 0.22 N/A 7.57 ± 0.40 26.97 ± 3.17
Non-compartment 3.0 ± 0.82 481.50 ± 44.55 3074.04 ± 356.03 0.24 ± 0.06 N/A N/A 2.98 ± 0.66 5.60 ± 0.83 7.03 ± 0.78 29.77 ± 4.25
Postnatal organs at P2W
Model Tmax (h) Cmax (g/kg) AUC (h g/kg) z (1/h) Kin (1/h) Kout (1/h) T1/2 (h) MRTinf (h) Cl/F (1/h) Vz /F
Kidney N = 55
Liver N = 55
Lung N = 55
Heart N = 55
Brain N = 55
Spleen N = 55
Non-compartment 5.50 ± 3.0* 10,854.30 ± 6654.84* 36,460.95 ± 9314.09* 0.25 ± 0.15 N/A N/A 3.54 ± 1.67 5.72 ± 1.76 0.62 ± 0.17* 3.18 ± 1.48*
Non-compartment 1.0 ± 0.61 2082.80 ± 435.45* 11,447.60 ± 1010.75* 0.21 ± 0.07 N/A N/A 3.66 ± 1.09 5.70 ± 1.43 1.88 ± 0.17* 9.90 ± 2.84*
Non-compartment 1.50 ± 1.0 2363.80 ± 1569.06 12,334.26 ± 3777.87* 0.21 ± 0.14 N/A N/A 4.67 ± 3.03 7.30 ± 4.22 1.85 ± 0.46* 11.42 ± 4.71*
Non-compartment 1.90 ± 1.82 1721.0 ± 521.10* 10,466.26 ± 973.57* 0.18 ± 0.06 N/A N/A 4.11 ± 1.09* 6.26 ± 1.49 2.06 ± 0.22* 12.12 ± 2.89*
Non-compartment 4.40 ± 1.14 1055.80 ± 126.05* 8054.82 ± 1673.91* 0.25 ± 0.19 N/A N/A 3.80 ± 1.81 7.57 ± 2.17 2.75 ± 0.54* 14.09 ± 5.10*
Non-compartment 1.13 ± 0.63 1920.0 ± 266.60 10,482.10 ± 2010.10 0.21 ± 0.07 N/A N/A 3.80 ± 1.86 5.98 ± 2.67 2.10 ± 0.40‡ 11.01 ± 3.74
n = 5 per time point, total N = 55 per group. Tmax , maximum peak concentration time of the prominent peak; Cmax , maximum concentration of the prominent peak; AUC, area under the curve integrate normalized to molarity; z , terminal slope or terminal elimination rate; Kin , first order constant for the production of response; Kout , first order rate constant for loss of response; T1/2 , half live (estimated from z and Kout in non-compartmental and 1-compartmental models, respectively); MRTinf , mean residence time from time zero to infinity; Cl/F, apparent clearance; and Vz /F, apparent volume of distribution. Data shows mean ± SD values. N/A, data not applicable. † Post hoc test: p < 0.05 comparing prenatal organs with prenatal kidney at E20 days. ‡ Post hoc test: p < 0.05 comparing postnatal organs with postnatal kidney at P2 weeks. * Post hoc test: p < 0.05 comparing the postnatal organ at P2 weeks with the same organ at prenatal E20 days.
pregnancy lower than those of in late pregnancy. The slowing down of the increase in body fluid volume in late pregnancy results in similar Tmax , Cmax and AUC to that of the non-pregnant rats. In our experiment, the estimated half-lives of melamine of the nonpregnant and the pregnant rat serum were 1.85–2.7 h. Significant difference was observed between non-pregnant rats and pregnant rats at E10, pregnant rats of E10 and E15, that of E15 and E20 as well as between pregnant rats of E10 and E20. Variation of T1/2 of the postnatal serum was bigger, from 1.29 to 4.26 h, the highest was observed in the rat of P2W. Significance was observed in rat at P2W against those at P6W, P8W and non-pregnant adult
and rat at P4W against those at P2W, P6W and non-pregnant adult. Result of pharmacokinetic analysis showed prenatal kidney at E20 did not differ much from that of other prenatal organs. Although prenatal rats start to produce urine in mid-gestation, development of kidney continues after birth. In this part of study about the different organs in prenatal and postnatal rats, prenatal liver showed the highest Cmax and AUC of melamine due to the large amount of blood flowing through the organ. Unfortunately, studies of the effect of melamine on liver cells are limited. There is only one report of a long term exposure of melamine to liver cell line L02,
Table 3 Pharmacokinetic parameters of melamine in prenatal and postnatal kidneys. Fetal kidney
Model Tmax (h) Cmax (g/kg) AUC (h g/kg) z (1/h) T1/2 (h) MRTinf (h) Cl/F (1/h) Vz /F
Postnatal kidney
E15 N = 55
E20 N = 55
P2W N = 55
P4W N = 55
P6W N = 55
P8W N = 55
Non-compartment 2.25 ± 0.96 184.68 ± 62.45 547.62 ± 42.40 0.31 ± 0.03 2.25 ± 0.24 4.18 ± 0.45 39.25 ± 3.0 127.86 ± 20.01
Non-compartment 2.20 ± 0.45 548.83 ± 70.32† 3029.02 ± 441.47† 0.23 ± 0.06 3.19 ± 0.92 5.15 ± 1.10 7.17 ± 0.93† 32.32 ± 7.01†
Non-compartment 5.50 ± 3.0 10,854.30 ± 6654.84 36,460.95 ± 9314.09 0.25 ± 0.15 3.54 ± 1.67 5.72 ± 1.76 0.62 ± 0.17 3.18 ± 1.48
Non-compartment 1.90 ± 0.89‡ 10,651.09 ± 8593.65 19,325.55 ± 9237.68‡ 0.73 ± 0.21‡ 1.047 ± 0.36‡ 2.44 ± 0.31‡ 1.30 ± 0.51‡ 2.10 ± 1.35
Non-compartment 1.0 ± 0.0‡ 4989.74 ± 764.75 16,013.15 ± 2950.61‡ 0.43 ± 0.12 1.77 ± 0.67 2.86 ± 0.62‡ 1.37 ± 0.21‡ 3.54 ± 1.58
Non-compartment 0.75 ± 0.29‡ 6365.29 ± 3496.91 13,847.34 ± 2547.83‡ 0.50 ± 0.10‡ 1.44 ± 0.26‡ 2.50 ± 0.58‡ 1.59 ± 0.34‡ 3.36 ± 1.24
n = 5 per time point, total N = 55 per group. Tmax , maximum peak concentration time of the prominent peak; Cmax , maximum concentration of the prominent peak; AUC, area under the curve integrate normalized to molarity; z , terminal slope or Terminal elimination rate; T1/2 , half live (estimated from z ); MRTinf , mean residence time from time zero to infinity; Cl/F, apparent clearance; and Vz /F, apparent volume of distribution. Data shows mean ± SD values. N/A, data not applicable. † Post hoc test: p < 0.05 for comparing prenatal E15 and E20 kidneys. ‡ Post hoc test: p < 0.05 comparing postnatal kidneys with the P2W kidney.
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Fig. 4. Tmax , Cmax and AUC of melamine in prenatal and postnatal organs. Prenatal organ samples (red bars) from gestational day 15 (E15) and day 20 (E20), N = 5 per time point, and postnatal organ samples (blue bars) from postnatal week 2 (P2W), N = 5 per time point, are shown. Mean are presented. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
but it did not demonstrate any effect of malignant transformation in vitro [14]. Tmax of melamine in prenatal brain at E20 was high. The small molecule of melamine may allow it to pass through the blood brain barrier and reach the brain while the re-distribution of melamine back into the circulation takes time. This is in agreement with the finding of our previous study, that melamine was able to pass the placental barrier and to be distributed to fetuses [8,12]. Moreover, the AUC of melamine to the prenatal brain was slightly higher than that of the prenatal kidney. Therefore, the effect of melamine on prenatal brain should not be overlooked. At postnatal 2 weeks of age, distribution of melamine became totally different. Distribution of melamine was much higher postnatal in kidney than in postnatal lung, liver, spleen, heart and brain. Tmax of melamine in postnatal brain was high, but it may
only be the result of presence of the blood–brain barrier. On the contrary, the total amount of melamine detected in postnatal brain was the lowest. From this observation, in the very young age of rats, kidney is accumulated with the highest amount of melamine. At early stage, postnatal kidneys pick up the role of ultrafiltration, control of water loss and removal of waste from the body. Crystals of melamine complex formed may easily block the delicate, yet not fully functional renal tubules, and causes lesion and scars to the kidneys. It is a general believe that melamine cannot form crystals alone, but has to be complex with its analogs. The presence of cyanuric acid, as a by-product of manufacture of melamine, is believed to be a co-contamination because it is not able to be converted from melamine in mammalian cells [6]. At room temperature, solubility of melamine and cyanuric acid are 3.2 g/L
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C.Y. Chu et al. / Reproductive Toxicology 35 (2013) 40–47
Fig. 5. Distribution of melamine in prenatal and postnatal kidneys. Prenatal kidneys (insert) from gestational day 15 (E15) and day 20 (E20), N = 5 per time point, and postnatal kidneys (main) from postnatal week 2 (P2W), week 4 (P4W), week 6 (P6W) and week 8 (P8W), N = 5 per time point, are shown. Mean ± SD are presented.
and 2.5 g/L, respectively; however, the solubility of the complex is almost zero. Due to the difference in their physical properties, the 2 compounds are believed to be absorbed separately in the acidic stomach (∼pH 2) and the basic small intestine (∼pH 8). Melamine cyanurate may then form in the neutral circulation (pH 7.4) and is concentrated in the slightly acidic urine (normal range of pH 4.4–8.2) [15]. However, other studies shown that melamine crystals are not necessarily formed with cyanuric acid, but uric acid [16]. Therefore, co-contamination by cyanuric acid is not essential for the formation of nephrolithiasis or urolithiasis in this health issue. After following up the children affected in the Chinese scandal of melamine, 2 groups of clinicians have reported the presence of cyanuric acid in the stones or the urine samples, while Sun’s group has detected the presence of uric acid in the melamine stones [16,17]. The distribution of melamine in postnatal kidney in P2W was more than double of the other organs of prenatal and postnatal rats. On day 12 of the rat fetal life, metanephric kidneys start to develop. Nephrogenesis continues in the first 3 weeks after birth. The average diameter and volume of developing glomeruli are found to remain the same in the rats younger than 5 weeks old while the glomerular size increases after 10 weeks [18]. This glomerular diameter allows the ultrafiltration rate to increase during kidney maturation. In our study, the Tmax of melamine in postnatal kidney at P2W was the highest, and so as the Cmax and AUC. The under-developed kidneys in these postnatal rats may have insufficient glomerular filtration and thus difficulties in removing melamine through urine excretion. This can make the neonates particularly vulnerable to the melamine nephrotoxicity. In human, kidneys will be fully functional by neonatal week 13 but they continue to grow for months. Melamine-tainted baby formulae or other dairy products are likely to cause damages to the delicate renal tubules of children under a year of age [19]. Fortunately, half-life of melamine is rather short. Half-life of melamine is estimated to be 3–4 h, in single bolus administration in pigs [5] or single daily administration in cats [20]. In this study, estimated half-lives of prenatal and postnatal kidney varied from
1.04 to 3.54 h. Significant difference of p < 0.05 was observed when comparing the half-life of kidney melamine of postnatal rat at P2W with those of postnatal rat at P4W and P8W. Half-lives of melamine in organs of rat at E20 were 2.47–3.19 h. Half-lives of melamine in organs of rat at P2W were slightly higher, ranging from 3.54 to 4.67 h. Only the heart showed significant difference of the halflives of melamine between the prenatal and the postnatal rats. However, the frequent intake of baby formula in early infancy may not provide enough time for the total clearance of the melamine between meals, causing gradual accumulation of the contaminant inside the body. Practically, after treatment like administration of fluid and alkalization of urine, milder cases can recover by increasing the solubility of melamine crystal in urine. Severe cases may need cystoscopy, retrograde catheterization, dialysis and ultrasound-guided extracorporeal shockwave lithotripsy in order to regain the normal renal function [16]. In recent reports about the melamine toxicity, melamine cyanurate crystals were found in kidney of rats fed with a mixture of the 2 components at 400 mg/kg body weight/day for 12 weeks [21]. Hyperplasia was found in kidney sections of rats received melamine alone at 6000 mg/kg body weight/day for 13 weeks [22]. Increased urinary bladder stones, hyperplasia and transitional-cell carcinoma were observed in rats received 4500 mg/kg body weight/day of melamine for 103 week [23]. In vitro studies using neuronal cells indicated melamine can induce apoptosis [24], hyperpolarization and spontaneous firing [25] and also induce deficit of learning and memory in rats [26]. These studies were all carried out on adult animals or cell models. Either toxicology studies or kinetic studies using pregnant animals are still lacking. By administrating 800 mg/kg body weight/day from gestational day 6 to 20, maternal rat kidneys increased in weight and incidences of renal tubular necrosis, crystals, dilation and recruitment of inflammatory cells. However, no histological abnormality was observed in fetal organs, except less calcification of fetal skeleton [27]. The link between distribution and toxicity of melamine in fetal organs requires further detailed studies. In the present study, the significant levels of melamine in prenatal kidney during late pregnancy and in postnatal
C.Y. Chu et al. / Reproductive Toxicology 35 (2013) 40–47
kidney during early postnatal period suggested the significant distribution of melamine in the developing kidneys. It may imply the high susceptibility of melamine toxicity observed in infants after exposure to the tainted infant formulae in early age [28]. 5. Conclusions We have characterized the change in distribution of the melamine in different stages of pregnancy and during the development of the prenatal and postnatal organs. During the early pregnancy, the distribution of the melamine after a single oral intake was less, but increased during the course of pregnancy. Toward the end of the pregnancy, the developing kidney was shown to have the similar distribution as the other organs studied. This risk of melamine toxicity to kidney may increase after birth as demonstrated by the tremendous increase in the distribution of melamine observed in the 2-week old rats. Since the development of rat fetus is less mature compared to the human fetus, fetal kidney of rat is comparable to that of human in the 2nd and the 3rd trimesters. As a consequence, the toxicity of melamine may have already posted to human fetuses in late pregnancy. Acknowledgement This study was supported by a funding on Studies Related to Melamine Incident from Food and Health Bureau, Hong Kong Special Administration Region (MI-BS-06). References [1] Dakin HD, Dudley HW. Some limitation of the Kjeldahl method. Journal of Biological Chemistry 1914;12:275–80. [2] Brown CA, Jeong KS, Poppenga RH, Puschner B, Miller DM, Ellis AE, et al. Outbreak of renal failure associated with melamine and cyanuric acid in dogs and cats in 2004 and 2007. Journal of Veterinary Diagnostic Investigation 2007;19:525–31. [3] Chen JS. A worldwide food safety concern in 2008—melamine-contaminated infant formula in China caused urinary tract stone in 290,000 children in China. Chinese Medical Journal 2009;122:243–4. [4] Gossner CME, Schlundt J, Embarek PB, Hird S, Lo-Fo-Wong D, Beltran JJO, et al. The melamine incident: implications for international food and feed safety. Environmental Health Perspectives 2009;117:1803–8. [5] Baynes RE, Smith G, Mason SE, Barrett E, Barlow BM, Riviere JE. Pharmacokinetics of melamine in pigs following intravenous administration. Food and Chemical Toxicology 2008;46:1196–200. [6] Panesar NS, Chan KW, Lo WS, Leung VH. Ho CS Co-contamination, but not mammalian cell conversion of melamine to cyanuric acid the likely cause of melamine-cyanurate nephrolithiasis. Clinica Chimica Acta 2010;411: 1830–1. [7] Wu YT, Huang CM, Lin CC, Ho WA, Lin LC, Chiu TF, et al. Determination of melamine in rat plasma, liver, kidney, spleen, bladder and brain by liquid chromatography–tandem mass spectrometry. Journal of Chromatography A 2009;1216:7595–601.
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[8] Chu CY, Chu KO, Chan JYW, Liu XZ, Ho CS, Wong CK, et al. Distribution of melamine in rat fetuses and neonates. Toxicology Letters 2010;199:398–402. [9] FDA. Interim safety and risk assessment of melamine and its analogues in food for humans [a]. http://www.fda.gov/Food/FoodSafety/ FoodContaminantsAdulteration/ChemicalContaminants/Melamine/ ucm164522.htm; 2008 [accessed 28.05.12]. [10] FDA. US Food and Drug Administration 2011 Dose Calculator. http://www.accessdata.fda.gov/scripts/cder/onctools/animalquery.cfm; [accessed 28.05.12]. [11] Chan JYW, Lau CM, Ting TL, Mak TCW, Chan MHM, Lam CWK, et al. Gestational and lactational transfer of melamine following gavage administration of a single dose to rats. Food and Chemical Toxicology 2011;49:1544–8. [12] CFS. Centre for Food Safety. Frequently asked questions. http://www. cfs.gov.hk/english/whatsnew/whatsnew fstr/whatsnew fstr Test dairy product FAQ.html; 2008 [accessed 28.05.12]. [13] Atherton JC, Pirie SC. The effect of pregnancy on glomerular filtration rate and salt and water reabsorption in the rat. Journal of Physiology 1981;319:153–64. [14] Zhang Q, Yang G, Li J, Li W, Zhang B, Zhu W. Melamine induces sperm DNA damage and abnormality, but not genetic toxicity. Regulatory Toxicology and Pharmacology 2011;60:144–50. [15] Varoe JS. Clinical biochemistry: techniques and instrumentation: a practical course. Singapore: World Scientific Publishing Co Ptc Ltd; 2001. pp. P5–9. [16] Sun Q, Shen Y, Sun N, Zhang GJ, Chen Z, Fan JF, et al. Diagnosis, treatment and follow-up of 25 patients with melamine-induced kidney stones complicated by acute obstructive renal failure in Beijing Children’s Hospital. European Journal of Pediatrics 2010;169:483–9. [17] Ding J. Childhood urinary stones induced by melamine-tainted formula: how much we know, how much we don’t know. Kidney International 2009;75:780–2. [18] Savin VJ, Beason-Griffin C, Richardson WP. Ultrafiltration coefficient of isolated glomeruli of rats aged 4 days to maturation. Kidney International 1985;28:926–31. [19] Chen WC, Wu SY, Liu HP, Chang CH, Chen HY, Chen HY, et al. Identification of melamine/cyanuric acid-containing nephrolithiasis by infrared spectroscopy. Journal of Clinical Laboratory Analysis 2010;24:92–9. [20] Puschner B, Poppenga RH, Lowenstine LJ, Filigenzi MS, Pesavento PA. Assessment of melamine and cyanuric acid toxicity in cats. Journal of Veterinary Diagnostic Investigation 2007;19:616–24. [21] Chen KC, Liao CW, Cheng FP, Chou CC, Chang SC, Wu JH, et al. Evaluation of subchronic toxicity of pet food contaminated with melamine and cyanuric acid in rats. Toxicologic Pathology 2009;37:959–68. [22] Hard GC, Flake GP, Sills RC. Re-evaluation of kidney histopathology from 13week toxicity and two-year carcinogenicity studies of melamine in the F344 rat: morphologic evidence of retrograde nephropathy. Veterinary Pathology 2009;46:1248–57. [23] Melnick RL, Boorman GA, Haseman JK, Montali RJ, Huff J. Urolithiasis and bladder carcinogenicity of melamine in rodents. Toxicology and Applied Pharmacology 1984;72:292–303. [24] Han YG, Liu SC, Zhang T, Yang Z. Induction of apoptosis by melamine in differentiated PC12 cells. Cellular and Molecular Neurobiology 2011;31: 65–71. [25] Yang J, An L, Yao Y, Yang Z, Zhang T. Melamine impairs spatial cognition and hippocampal synaptic plasticity by presynaptic inhibition of glutamatergic transmission in infant rats. Toxicology 2011;289:167–74. [26] An L, Li Z, Yang Z, Zhang T. Cognitive deficits induced by melamine in rats. Toxicology Letters 2011;206:276–80. [27] Kim SH, Lee IC, Lim JH, Shin IS, Moon C, Kim SH, et al. Effect of melamine on pregnant dams and embryo-fetal development in rats. Journal of Applied Toxicology 2011, http://dx.doi.org/10.1002/jat.1703. [28] Zhang L, Wu LL, Wang YP, Liu AM, Zou CC, Zhao ZY. Melamine-contaminated milk products induced urinary tract calculi in children. World Journal of Pediatrics 2009;5:31–5.
Contents lists available at SciVerse ScienceDirect
Reproductive Toxicology journal homepage: www.elsevier.com/locate/reprotox
Melamine in prenatal and postnatal organs in rats Ching Yan Chu a , Kai On Chu a,b , Chung Shun Ho c , Sung Shing Kwok c , Ho Ming Chan c , Kwok Pui Fung d , Chi Chiu Wang a,d,∗ a
Department of Obstetrics and Gynaecology, Hong Kong Department of Ophthalmology and Visual Sciences, The Chinese University of Hong Kong, Hong Kong Department of Chemical Pathology, Prince of Wales Hospital, Hong Kong d School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong b c
a r t i c l e
i n f o
Article history: Received 24 February 2012 Received in revised form 5 June 2012 Accepted 15 June 2012 Available online 23 June 2012 Keywords: Melamine Pharmacokinetics Distribution Pregnancy Infants
a b s t r a c t Melamine can be transferred to fetus in utero through placenta and to infant ex utero by breast feeding. In this study, we characterized the pharmacokinetics of melamine in prenatal and postnatal organs in rats. Single bolus of melamine was administered to pregnant rats at different gestational stages and to infants at different postnatal stages. Distribution of melamine in maternal serum was about 30% higher in late pregnancy than that in early pregnancy; and it was 2 folds higher in postnatal serum in early infants in young adulthood. Distribution of melamine in all postnatal organs was higher than that in prenatal organs. Postnatal kidneys in early infants had the highest maximum concentration and the lowest clearance of melamine than the other postnatal organs. It may relate to the high vulnerability to the toxicity of melamine in this population. © 2012 Elsevier Inc. All rights reserved.
1. Introduction Nephrotoxic melamine (2,4,6-triamino-1,3,5-triazine) has been contaminating food for years. It was introduced to food sources as one of the adulteration with high nitrogen content in dairy products. The intention was to deceive the routine tests like Kjeldahl reaction [1] that cannot distinguish nitrogen atoms of a proteinaceous source from the others, such as amides, ammonium, alkaloids, urea, as well as melamine. The deliberate adulteration of pet food and infant formula by melamine, resulting in death of pets
Abbreviations: ACN, acetonitrile; AUC, area under the curve; AUMC, predicted area under the first moment curve divided by the predicted AUC; Cl/F, apparent oral clearance; Cmax , maximum concentration; FDA, The US Food and Drug Administration; GFR, glomerular filtration rate; Kin , absorption rate constant; Kout , elimination rate constant; z , terminal elimination rate; LC–MS, liquid chromatography–mass spectrometry; MRTinf , mean residence time extrapolated to infinity; T1/2 , half-life; Tmax , time of maximum concentration; Vz /F, apparent volume of distribution. ∗ Corresponding author at: Department of Obstetrics and Gynaecology, The Chinese University of Hong Kong, Shatin, Hong Kong. Tel.: +852 2632 2810; fax: +852 2636 0008. E-mail addresses: nina [email protected] (C.Y. Chu), [email protected] (K.O. Chu), [email protected] (C.S. Ho), [email protected] (S.S. Kwok), [email protected] (H.M. Chan), [email protected] (K.P. Fung), [email protected] (C.C. Wang). 0890-6238/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.reprotox.2012.06.003
and acute renal failure in human, has raised immense concern on the safety issue worldwide [2–4]. Melamine is not metabolized in human body. It is excreted in urine per se [5,6]. It has low direct toxicity to human but its low solubility is believed to be the culprit of its damage to kidneys. When melamine forms a complex with cyanuric acid, which is almost undissolvable in water, it easily blocks the renal tubules [2]. A communiqué in 2008 stated that tens of thousands of children were hospitalized after taking in the adulterated baby formula in the People Republic of China. Most of the affected children have been diagnosed with anuria, kidney stones, urinary tract stones, hydronephrosis, even more severe cases with acute renal failure and death were reported [3]. Besides kidney, melamine was also detected in other tissues, e.g., liver, spleen, bladder and brain [7]. Developing organs in fetus in utero and neonates ex utero are highly susceptible to the melamine accumulation [8]. In this study, we would like to investigate the detailed pharmacokinetics of melamine in prenatal and postnatal organs at different developmental stages. 2. Method 2.1. Chemicals Melamine for animal experiments and solvents for LC–MS analytical measurement were purchased from Sigma Chemical Co (St. Louis, MO). 13 C3 ,15 N3 -melamine (Cambridge Isotope Laboratories, Andover, MA) was used as the internal standard.
C.Y. Chu et al. / Reproductive Toxicology 35 (2013) 40–47 2.2. Animals In order to determine the pharmacokinetic of melamine in vivo, pregnant and postnatal Sprague-Dawley rats were used. Animals were obtained from the Laboratory Animal Services Center of the Chinese University of Hong Kong. Ethics approval for the use and care of animal study was obtained from the Animal Ethics Committee of the University. All animals were housed at 25 ◦ C with a 12-h light/dark cycle with free access of standard water and chow. Young pups were housed with lactating rats before weaning.
2.3. Dosage Both the prenatal and the postnatal rats were investigated. All animals, except lactating rats, received a single oral bolus of melamine at 21.4 mg/kg of body weight as previously described [8]. The dosage for rat was chosen as 5.5 fold of the equivalent dose of human tolerable daily intake (TDI) of melamine recommended by the US Food and Drug Administration (FDA) in 2008 [9] and converted by the online FDA Dose Calculator [10]. The dosage conversion from human dose to rat dose included height, weight and surface area and was corrected by conversion factor, i.e. 6, as previously described [11]. The selected dose is also equivalent to the melamine levels detected in the contaminated dairy food products in the local area [12].
2.4. Administration Pregnant rats at the gestational day 10, 15 and 20 (E10, E15 and E20) and infant rats at postnatal week 2, 4, 6 and 8 (P2W, P4W, P6W and P8W) were weight and gavaged with melamine in distilled water. Lactating mothers were not administered the melamine during the experiments for postnatal pups. All animals were sacrificed and samples were collected at 0, 0.5, 1, 2, 3, 4, 5, 6, 7, 8 and 24 h after administration. Maternal and infant serum were prepared from peripheral whole blood from inferior vena cavae at all gestational and postnatal periods, except decapitation for the 2-week old neonates. Serum were centrifuged at 3000 rpm, at 4 ◦ C for 10 min. Fetal kidneys at E15 and, fetal kidneys, livers, lungs, hearts and brains at E20 were collected. Each of the prenatal organs of the same litter was pooled together as a single sample. Postnatal kidneys were collected in all the postnatal periods, while postnatal livers, lungs, hearts, spleens and brains were also collected in 2-week old rats. All tissues were washed in PBS and all samples were frozen at −80 ◦ C prior to preparation for melamine quantitation by LC–MS. N = 5 for each time point, i.e. total sample number of animals for each developmental stage were 55.
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The half-lives (T1/2 ) of melamine in various samples were estimated by the z and the Kout , the product of ln 2 and the reciprocal of z or Kout for experimental comparison. T1/2 =
ln 2 z
or
T1/2 =
ln 2 z
2.7. Statistical analysis Statistical analysis of Tmax , Cmax , z , T1/2 , MRTinf , Cl/F and Vz /F of the maternal serum of the pregnant rats versus that of the non-pregnant adult control and the infant serum at other stages versus that at P2W were carried out by one-way ANOVA. T-tests assuming equal variants (2-tailed) were followed when there was significance detected by ANOVA. One-way ANOVA was also applied to organs of the same prenatal and postnatal stages on Tmax , Cmax , and AUC. Pharmacokinetic parameters between the prenatal and the postnatal kidneys were compared by T-test.
3. Results 3.1. Maternal and postnatal serum All maternal and postnatal serum followed the noncompartmental pharmacokinetic model. Single peak was obtained from the melamine distribution over the 24-h period. The pharmacokinetic parameters were shown in Table 1. The pharmacokinetic profiles in maternal serum were similar in non-pregnant and pregnant rat at E20, but different from those of E10 and E15. The pharmacokinetic profiles of postnatal serum at P4W, P6W and P8W were similar, but not to that of P2W rats (Fig. 1). An
2.5. Quantitation of melamine Acetonitrile (ACN)/water (50:50, v/v) was used for tissue homogenization and melamine extraction from tissues. Sera required no extraction. Melamine in both tissue homogenates and sera were analyzed by UPLC-MS Waters Xevo TQ System (Waters, Milford, MA). Extraction and measurement of melamine were performed as described previously [8]. The limit of quantitation was 5 ppb and the linearity was up to 10,000 ppb. The recovery of the assay was 105 ± 13%.
2.6. Pharmacokinetic analysis Pharmacokinetic analysis was carried out by WINNONLIN Professional Version 4.01 (Pharsight Corp, MO). Maximum concentration (Cmax ), time of Cmax (Tmax ) and area under the curve (AUC) of melamine in each sample was computed. Exposure of melamine was presented as normalized AUC calculated by the trapezoidal formula in terms of ng/g h. The average time of melamine staying in the samples from zero time point to infinity (mean residence time extrapolated to infinity, MRTinf ) was obtained from the predicted area under the first moment curve divided by the predicted AUC (AUMC) from zero time point to infinity. MRTinf =
AUMCinf AUCinf
The overall elimination rate of the melamine calculated from the terminal slope was expressed as terminal elimination rate (z ) which described the elimination rate of the molecule following non-compartment model. Absorption rate constant (Kin ) as the rate constant for the oral melamine intake and elimination rate (Kout ) as the elimination rate constant of melamine were obtained from first order model. Apparent oral clearance (Cl/F) and apparent volume of distribution (Vz /F) were determined from the terminal slope. Cl/F =
Dose AUCinf
Vz /F =
Dose z × AUCinf
Fig. 1. Distribution of melamine in maternal and postnatal sera. Maternal serum samples (upper panel) from non-pregnant and pregnant rats at gestational day 10 (E10), day 15 (E15) and day 20 (E20), N = 5 per time point and postnatal serum samples (lower panel) from postnatal rats at 2 weeks (P2W), 4 weeks (P4W), 6 weeks (P6W) and 8 weeks (P8W), N = 5 per time point, are shown. Mean ± SD are presented.
n = 5 per time point, total N = 55 per group. Tmax , maximum peak concentration time of the prominent peak; Cmax , maximum concentration of the prominent peak; AUC, area under the curve integrate normalized to molarity; z , terminal slope or terminal elimination rate; T1/2 , half live (estimated from z ); MRTinf , mean residence time from time zero to infinity; Cl/F, apparent clearance; and Vz /F, apparent volume of distribution. Data shows mean ± SD values. N/A, data not applicable. † Post hoc test: p < 0.05 comparing maternal serum with non-pregnant rats serum. ‡ Post hoc test: p < 0.05 comparing postnatal sera with the P2W serum.
N = 55 N = 55
Non-compartment 0.88 ± 0.25 10,315.75 ± 546.34 32,838.17 ± 1824.92‡ 0.32 ± 0.09‡ 2.29 ± 0.52‡ 2.76 ± 0.17‡ 0.65 ± 0.04 2.16 ± 0.50
N = 55
Non-compartment 0.8 ± 0.27 9911.80 ± 1881.75 26,695.55 ± 1603.62‡ 0.54 ± 0.06‡ 1.29 ± 0.13‡ 2.33 ± 0.21‡ 0.80 ± 0.05 1.51 ± 0.23
N = 55
Non-compartment 1.80 ± 1.89 12,242.80 ± 3112.51 72,177.15 ± 7004.65 0.17 ± 0.05 4.26 ± 1.15 6.53 ± 1.72 0.30 ± 0.03 1.82 ± 0.46
N = 55 N = 55 N = 55
Non-compartment 0.90 ± 0.22 10,609.80 ± 1685.23† 34,860.0 ± 2291.41† 0.38 ± 0.03† 1.85 ± 0.13† 3.0 ± 0.22 0.62 ± 0.04† 1.64 ± 0.13 Non-compartment 0.75 ± 0.29 14,465.25 ± 1380.63 50,975.13 ± 1849.49 0.28 ± 0.03 2.55 ± 0.34 3.32 ± 0.27 0.42 ± 0.02 1.55 ± 0.22 Model Tmax (h) Cmax (g/kg) AUC (h g/kg) z (1/h) T1/2 (h) MRTinf (h) Cl/F (1/h) Vz /F
N = 55
Non-compartment 1.20 ± 1.04 11,581.20 ± 2700.51 39,138.43 ± 2809.09† 0.26 ± 0.02 2.70 ± 0.18 3.34 ± 0.34 0.55 ± 0.04† 2.14 ± 0.23†
Non-compartment 1.0 ± 0.0 14,308.0 ± 1471.76 51,447.58 ± 2160.24 0.32 ± 0.04 2.19 ± 0.24 3.53 ± 0.33 0.42 ± 0.02 1.32 ± 0.16
P6W P4W Postnatal Serum
P2W E20 E15 E10 Non-pregnant
Maternal serum
Table 1 Pharmacokinetic parameters of melamine in maternal and postnatal sera.
Non-compartment 0.88 ± 0.25 11,355.50 ± 3185.49 34,483.97 ± 8664.15‡ 0.35 ± 0.09‡ 2.08 ± 0.52‡ 2.96 ± 0.19‡ 0.65 ± 0.15 1.89 ± 0.38
C.Y. Chu et al. / Reproductive Toxicology 35 (2013) 40–47
P8W
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Fig. 2. Tmax , Cmax and AUC of melamine in maternal and postnatal sera. Maternal serum samples (red bars) from non-pregnant and pregnant rats at gestational day 10 (E10), day 15 (E15) and day 20 (E20), per time point, and postnatal serum samples (blue bars) from postnatal rats at 2 week (P2W), 4 weeks (P4W), 6 weeks (P6W) and 8 weeks (P8W), N = 5 per time point, are shown. Mean are presented. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
increase in Tmax of maternal serum melamine was observed from the non-pregnant to the pregnant rat at E15 (1.6 times of that of that non-pregnant rat), and then dropped at E20 (1.3 times) (Fig. 2). Postnatal serum had the highest Tmax of melamine at P2W, and then decreased by half and maintained at this level for the following 6 weeks (0.44–0.48 times). Comparing the Tmax of serum melamine of P8W and that of the non-pregnant adult rat, there was a further but slight drop observed, with no significant difference. Cmax of serum melamine of non-pregnant rat was the highest (Fig. 2). During the course of pregnancy, Cmax of melamine rose; in late pregnancy, Cmax became comparable to that of the non-pregnant one. Cmax of melamine of maternal serum were 0.73, 0.80 and 0.99 fold at E10, E15 and E20, compared to that of the non-pregnant ones. Cmax of serum melamine of pregnant rat at E10 showed a significant difference to the non-pregnant rat and the non-pregnant rats at E20 (p < 0.01). Cmax of melamine of postnatal serum became the highest at P2W. Cmax of melamine of rats at P4W, P6W and P8W were 0.81, 0.84 and 0.93 fold of that of rat at P2W, respectively. Similar trends were obtained in the AUC of serum melamine of the neonates (0.37–0.48 fold of that of rat at postnatal week 2) (Fig. 2). AUC of serum melamine of postnatal
C.Y. Chu et al. / Reproductive Toxicology 35 (2013) 40–47
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followed by prenatal kidney, lung, heart and liver (Fig. 4). The Tmax of melamine of these prenatal organs were 0.73, 0.42, 0.40 and 0.39 times of that of prenatal brain, respectively. Pharmacokinetic analysis of melamine in prenatal liver showed the highest Cmax and AUC (Fig. 4). Cmax of melamine of prenatal organs in descending order were liver, heart (0.82 fold), lung (0.80 fold), kidney (0.75 fold) and brain (0.65 fold of that of liver); AUC of melamine of prenatal organs in descending order were liver, brain (0.85 fold), kidney (0.84 fold), lung (0.83 fold), and heart (0.78 fold of that of liver). Melamine measured in the postnatal kidney at P2W had the highest Tmax , Cmax and AUC among all other postnatal organs collected (Fig. 4). Tmax of melamine in postnatal brain was also high, i.e., 0.8 fold of that of postnatal kidney. On the other hand, peaks of the melamine profile of the other postnatal organs were observed much earlier in postnatal heart (0.35 times), lung (0.27 times), spleen (0.20 times) and liver (0.18 times of kidney), in descending order. Despite of the high Tmax of melamine in postnatal brain, the other 2 parameters, Cmax and AUC of melamine in postnatal brain were comparable to those of the other organs (Fig. 4). Cmax and AUC of melamine in postnatal kidney were particularly high. Cmax of melamine in postnatal lung, liver, spleen, heart and brain of rat at P2W were only 0.22, 0.19, 0.18, 0.15 and 0.10 of that of postnatal kidney, respectively. AUC of melamine had the same descending order of that of Cmax . Those of postnatal lung, liver, spleen, heart and brain were observed to be only 0.34, 0.31, 0.29, 0.29 and 0.22 of that of postnatal kidney. Tmax of melamine in postnatal kidney at P2W (p < 0.05), Cmax of melamine in postnatal liver, heart and brain (p < 0.005) and postnatal kidney (p < 0.01), and AUC of all postnatal organs (p < 0.005) were significantly higher than that in the prenatal organs at E20. 3.3. Prenatal and postnatal kidneys
rats at 2 week was significantly different from that of the other 3 postnatal groups as well as from that of the non-pregnant adult (p < 0.005). Non-pregnant adult also showed a very significantly different AUC of serum melamine from that of the postnatal rats (p < 0.005), except those of P8W (p < 0.01). AUC of serum melamine of the pregnant adults increased gradually throughout the pregnancy. In the later stage of pregnancy, the AUC of serum melamine was similar to that of the non-pregnant adult (0.68–1.01 fold of that of the non-pregnant rat, from E10 to E20). AUC of serum melamine of both of the non-pregnant rats and the pregnant rats at E20 day showed a big significant difference against that of the pregnant rats at earlier stages (p < 0.005).
Since distinct pharmacokinetic profiles of melamine in prenatal and postnatal kidneys were observed, pharmacokinetic properties in the developing kidneys between E20 and P2W were further compared (Fig. 5 and Table 3). Both the Cmax and the AUC of melamine in prenatal kidneys significantly lower than that in postnatal kidneys (p < 0.005). Tmax and AUC of melamine in postnatal kidneys were the highest (Fig. 4). Tmax of melamine of kidneys at P4W, P6W and P8W were detected much earlier, only 0.35, 0.18 and 0.14 times of that of rat at P2W; also AUC of melamine of kidneys at P4W, P6W and P8W only 0.53, 0.44 and 0.38 fold of that of rat at P2W, respectively (Fig. 4). Tmax of melamine in kidneys of postnatal rat at P2W is significantly higher from the other postnatal groups (p < 0.05); AUC of melamine in kidney at P2W is also significantly higher from that at P6W and P8W (p < 0.005). Cmax displayed a different trend, having that of the rats at week 2 and 4 higher than the rest, and that of rats of P6W the lowest (Fig. 4). Cmax of melamine of rats at P4W, P6W and P8W were 0.98, 0.46 and 0.59 fold of that of rat at P2W, respectively.
3.2. Prenatal and postnatal organs
4. Discussion
Distribution of melamine in maternal serum was the highest in E20 and so as that of in the postnatal serum at P2W. Further pharmacokinetic comparison in prenatal and postnatal organs was done between these 2 developmental stages. Pharmacokinetics of melamine of prenatal liver, lung and heart at E20 followed 1compartment model while the other prenatal organs of the same stage, i.e., kidney and brain, as well as those of the postnatal rats at 2 week, followed the non-compartmental model. In each of the profiles, only 1 peak was observed (Fig. 3). Pharmacokinetic parameters are shown in Table 2. Tmax of melamine of prenatal brain of E20 rats was the highest among the 5 prenatal organs we had collected,
During the course of pregnancy, maternal blood volume increases. Glomerular filtration rate (GFR), salt and water reabsorption increase dramatically during the early pregnancy and last until at least mid-pregnancy. Therefore, volume of body fluid increases, most rapidly during the mid-pregnancy. Toward the later stage of pregnancy, GFR decreases slightly while the reabsorption of salt and water continue to be high [13]. As a consequence, more melamine can be excreted by the maternal kidney, especially during the early and the mid-pregnancy. The dilution effect, or change in distribution of maternal melamine, reflected by the pharmacokinetic parameters Cmax and AUC of the pregnant rats in early stages of
Fig. 3. Distribution of melamine in prenatal and postnatal organs. Prenatal organs (upper panel), N = 5 per time point, from gestational day 20 and postnatal organs (lower panel), N = 5 per time point, from postnatal week 2 are shown. Mean ± SD are presented.
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Table 2 Pharmacokinetic parameters of melamine in prenatal organs and postnatal organs. Fetal organs at E20
Model Tmax (h) Cmax (g/kg) AUC (h g/kg) z (1/h) Kin (1/h) Kout (1/h) T1/2 (h) MRTinf (h) Cl/F (1/h) Vz /F
Kidney N = 55
Liver N = 55
Lung N = 55
Heart N = 55
Brain N = 55
Non-compartment 2.20 ± 0.45 548.83 ± 70.32 3029.02 ± 441.47 0.23 ± 0.06 N/A N/A 3.19 ± 0.92 5.15 ± 1.10 7.17 ± 0.93 32.32 ± 7.01
1-Compartment 1.18 ± 0.26† 735.70 ± 86.46† 3611.24 ± 420.28 N/A 3.95 ± 2.25 0.26 ± 0.05 2.74 ± 0.42 N/A 5.98 ± 0.64 23.68 ± 4.64
1-Compartment 1.25 ± 0.22† 589.12 ± 50.61 2980.0 ± 159.24 N/A 3.09 ± 1.31 0.25 ± 0.03 2.77 ± 0.31 N/A 7.20 ± 0.37 28.87 ± 4.17
1-Compartment 1.21 ± 0.34† 600.87 ± 17.77 2833.29 ± 147.95 N/A 2.82 ± 1.44 0.28 ± 0.03 2.47 ± 0.22 N/A 7.57 ± 0.40 26.97 ± 3.17
Non-compartment 3.0 ± 0.82 481.50 ± 44.55 3074.04 ± 356.03 0.24 ± 0.06 N/A N/A 2.98 ± 0.66 5.60 ± 0.83 7.03 ± 0.78 29.77 ± 4.25
Postnatal organs at P2W
Model Tmax (h) Cmax (g/kg) AUC (h g/kg) z (1/h) Kin (1/h) Kout (1/h) T1/2 (h) MRTinf (h) Cl/F (1/h) Vz /F
Kidney N = 55
Liver N = 55
Lung N = 55
Heart N = 55
Brain N = 55
Spleen N = 55
Non-compartment 5.50 ± 3.0* 10,854.30 ± 6654.84* 36,460.95 ± 9314.09* 0.25 ± 0.15 N/A N/A 3.54 ± 1.67 5.72 ± 1.76 0.62 ± 0.17* 3.18 ± 1.48*
Non-compartment 1.0 ± 0.61 2082.80 ± 435.45* 11,447.60 ± 1010.75* 0.21 ± 0.07 N/A N/A 3.66 ± 1.09 5.70 ± 1.43 1.88 ± 0.17* 9.90 ± 2.84*
Non-compartment 1.50 ± 1.0 2363.80 ± 1569.06 12,334.26 ± 3777.87* 0.21 ± 0.14 N/A N/A 4.67 ± 3.03 7.30 ± 4.22 1.85 ± 0.46* 11.42 ± 4.71*
Non-compartment 1.90 ± 1.82 1721.0 ± 521.10* 10,466.26 ± 973.57* 0.18 ± 0.06 N/A N/A 4.11 ± 1.09* 6.26 ± 1.49 2.06 ± 0.22* 12.12 ± 2.89*
Non-compartment 4.40 ± 1.14 1055.80 ± 126.05* 8054.82 ± 1673.91* 0.25 ± 0.19 N/A N/A 3.80 ± 1.81 7.57 ± 2.17 2.75 ± 0.54* 14.09 ± 5.10*
Non-compartment 1.13 ± 0.63 1920.0 ± 266.60 10,482.10 ± 2010.10 0.21 ± 0.07 N/A N/A 3.80 ± 1.86 5.98 ± 2.67 2.10 ± 0.40‡ 11.01 ± 3.74
n = 5 per time point, total N = 55 per group. Tmax , maximum peak concentration time of the prominent peak; Cmax , maximum concentration of the prominent peak; AUC, area under the curve integrate normalized to molarity; z , terminal slope or terminal elimination rate; Kin , first order constant for the production of response; Kout , first order rate constant for loss of response; T1/2 , half live (estimated from z and Kout in non-compartmental and 1-compartmental models, respectively); MRTinf , mean residence time from time zero to infinity; Cl/F, apparent clearance; and Vz /F, apparent volume of distribution. Data shows mean ± SD values. N/A, data not applicable. † Post hoc test: p < 0.05 comparing prenatal organs with prenatal kidney at E20 days. ‡ Post hoc test: p < 0.05 comparing postnatal organs with postnatal kidney at P2 weeks. * Post hoc test: p < 0.05 comparing the postnatal organ at P2 weeks with the same organ at prenatal E20 days.
pregnancy lower than those of in late pregnancy. The slowing down of the increase in body fluid volume in late pregnancy results in similar Tmax , Cmax and AUC to that of the non-pregnant rats. In our experiment, the estimated half-lives of melamine of the nonpregnant and the pregnant rat serum were 1.85–2.7 h. Significant difference was observed between non-pregnant rats and pregnant rats at E10, pregnant rats of E10 and E15, that of E15 and E20 as well as between pregnant rats of E10 and E20. Variation of T1/2 of the postnatal serum was bigger, from 1.29 to 4.26 h, the highest was observed in the rat of P2W. Significance was observed in rat at P2W against those at P6W, P8W and non-pregnant adult
and rat at P4W against those at P2W, P6W and non-pregnant adult. Result of pharmacokinetic analysis showed prenatal kidney at E20 did not differ much from that of other prenatal organs. Although prenatal rats start to produce urine in mid-gestation, development of kidney continues after birth. In this part of study about the different organs in prenatal and postnatal rats, prenatal liver showed the highest Cmax and AUC of melamine due to the large amount of blood flowing through the organ. Unfortunately, studies of the effect of melamine on liver cells are limited. There is only one report of a long term exposure of melamine to liver cell line L02,
Table 3 Pharmacokinetic parameters of melamine in prenatal and postnatal kidneys. Fetal kidney
Model Tmax (h) Cmax (g/kg) AUC (h g/kg) z (1/h) T1/2 (h) MRTinf (h) Cl/F (1/h) Vz /F
Postnatal kidney
E15 N = 55
E20 N = 55
P2W N = 55
P4W N = 55
P6W N = 55
P8W N = 55
Non-compartment 2.25 ± 0.96 184.68 ± 62.45 547.62 ± 42.40 0.31 ± 0.03 2.25 ± 0.24 4.18 ± 0.45 39.25 ± 3.0 127.86 ± 20.01
Non-compartment 2.20 ± 0.45 548.83 ± 70.32† 3029.02 ± 441.47† 0.23 ± 0.06 3.19 ± 0.92 5.15 ± 1.10 7.17 ± 0.93† 32.32 ± 7.01†
Non-compartment 5.50 ± 3.0 10,854.30 ± 6654.84 36,460.95 ± 9314.09 0.25 ± 0.15 3.54 ± 1.67 5.72 ± 1.76 0.62 ± 0.17 3.18 ± 1.48
Non-compartment 1.90 ± 0.89‡ 10,651.09 ± 8593.65 19,325.55 ± 9237.68‡ 0.73 ± 0.21‡ 1.047 ± 0.36‡ 2.44 ± 0.31‡ 1.30 ± 0.51‡ 2.10 ± 1.35
Non-compartment 1.0 ± 0.0‡ 4989.74 ± 764.75 16,013.15 ± 2950.61‡ 0.43 ± 0.12 1.77 ± 0.67 2.86 ± 0.62‡ 1.37 ± 0.21‡ 3.54 ± 1.58
Non-compartment 0.75 ± 0.29‡ 6365.29 ± 3496.91 13,847.34 ± 2547.83‡ 0.50 ± 0.10‡ 1.44 ± 0.26‡ 2.50 ± 0.58‡ 1.59 ± 0.34‡ 3.36 ± 1.24
n = 5 per time point, total N = 55 per group. Tmax , maximum peak concentration time of the prominent peak; Cmax , maximum concentration of the prominent peak; AUC, area under the curve integrate normalized to molarity; z , terminal slope or Terminal elimination rate; T1/2 , half live (estimated from z ); MRTinf , mean residence time from time zero to infinity; Cl/F, apparent clearance; and Vz /F, apparent volume of distribution. Data shows mean ± SD values. N/A, data not applicable. † Post hoc test: p < 0.05 for comparing prenatal E15 and E20 kidneys. ‡ Post hoc test: p < 0.05 comparing postnatal kidneys with the P2W kidney.
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Fig. 4. Tmax , Cmax and AUC of melamine in prenatal and postnatal organs. Prenatal organ samples (red bars) from gestational day 15 (E15) and day 20 (E20), N = 5 per time point, and postnatal organ samples (blue bars) from postnatal week 2 (P2W), N = 5 per time point, are shown. Mean are presented. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
but it did not demonstrate any effect of malignant transformation in vitro [14]. Tmax of melamine in prenatal brain at E20 was high. The small molecule of melamine may allow it to pass through the blood brain barrier and reach the brain while the re-distribution of melamine back into the circulation takes time. This is in agreement with the finding of our previous study, that melamine was able to pass the placental barrier and to be distributed to fetuses [8,12]. Moreover, the AUC of melamine to the prenatal brain was slightly higher than that of the prenatal kidney. Therefore, the effect of melamine on prenatal brain should not be overlooked. At postnatal 2 weeks of age, distribution of melamine became totally different. Distribution of melamine was much higher postnatal in kidney than in postnatal lung, liver, spleen, heart and brain. Tmax of melamine in postnatal brain was high, but it may
only be the result of presence of the blood–brain barrier. On the contrary, the total amount of melamine detected in postnatal brain was the lowest. From this observation, in the very young age of rats, kidney is accumulated with the highest amount of melamine. At early stage, postnatal kidneys pick up the role of ultrafiltration, control of water loss and removal of waste from the body. Crystals of melamine complex formed may easily block the delicate, yet not fully functional renal tubules, and causes lesion and scars to the kidneys. It is a general believe that melamine cannot form crystals alone, but has to be complex with its analogs. The presence of cyanuric acid, as a by-product of manufacture of melamine, is believed to be a co-contamination because it is not able to be converted from melamine in mammalian cells [6]. At room temperature, solubility of melamine and cyanuric acid are 3.2 g/L
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Fig. 5. Distribution of melamine in prenatal and postnatal kidneys. Prenatal kidneys (insert) from gestational day 15 (E15) and day 20 (E20), N = 5 per time point, and postnatal kidneys (main) from postnatal week 2 (P2W), week 4 (P4W), week 6 (P6W) and week 8 (P8W), N = 5 per time point, are shown. Mean ± SD are presented.
and 2.5 g/L, respectively; however, the solubility of the complex is almost zero. Due to the difference in their physical properties, the 2 compounds are believed to be absorbed separately in the acidic stomach (∼pH 2) and the basic small intestine (∼pH 8). Melamine cyanurate may then form in the neutral circulation (pH 7.4) and is concentrated in the slightly acidic urine (normal range of pH 4.4–8.2) [15]. However, other studies shown that melamine crystals are not necessarily formed with cyanuric acid, but uric acid [16]. Therefore, co-contamination by cyanuric acid is not essential for the formation of nephrolithiasis or urolithiasis in this health issue. After following up the children affected in the Chinese scandal of melamine, 2 groups of clinicians have reported the presence of cyanuric acid in the stones or the urine samples, while Sun’s group has detected the presence of uric acid in the melamine stones [16,17]. The distribution of melamine in postnatal kidney in P2W was more than double of the other organs of prenatal and postnatal rats. On day 12 of the rat fetal life, metanephric kidneys start to develop. Nephrogenesis continues in the first 3 weeks after birth. The average diameter and volume of developing glomeruli are found to remain the same in the rats younger than 5 weeks old while the glomerular size increases after 10 weeks [18]. This glomerular diameter allows the ultrafiltration rate to increase during kidney maturation. In our study, the Tmax of melamine in postnatal kidney at P2W was the highest, and so as the Cmax and AUC. The under-developed kidneys in these postnatal rats may have insufficient glomerular filtration and thus difficulties in removing melamine through urine excretion. This can make the neonates particularly vulnerable to the melamine nephrotoxicity. In human, kidneys will be fully functional by neonatal week 13 but they continue to grow for months. Melamine-tainted baby formulae or other dairy products are likely to cause damages to the delicate renal tubules of children under a year of age [19]. Fortunately, half-life of melamine is rather short. Half-life of melamine is estimated to be 3–4 h, in single bolus administration in pigs [5] or single daily administration in cats [20]. In this study, estimated half-lives of prenatal and postnatal kidney varied from
1.04 to 3.54 h. Significant difference of p < 0.05 was observed when comparing the half-life of kidney melamine of postnatal rat at P2W with those of postnatal rat at P4W and P8W. Half-lives of melamine in organs of rat at E20 were 2.47–3.19 h. Half-lives of melamine in organs of rat at P2W were slightly higher, ranging from 3.54 to 4.67 h. Only the heart showed significant difference of the halflives of melamine between the prenatal and the postnatal rats. However, the frequent intake of baby formula in early infancy may not provide enough time for the total clearance of the melamine between meals, causing gradual accumulation of the contaminant inside the body. Practically, after treatment like administration of fluid and alkalization of urine, milder cases can recover by increasing the solubility of melamine crystal in urine. Severe cases may need cystoscopy, retrograde catheterization, dialysis and ultrasound-guided extracorporeal shockwave lithotripsy in order to regain the normal renal function [16]. In recent reports about the melamine toxicity, melamine cyanurate crystals were found in kidney of rats fed with a mixture of the 2 components at 400 mg/kg body weight/day for 12 weeks [21]. Hyperplasia was found in kidney sections of rats received melamine alone at 6000 mg/kg body weight/day for 13 weeks [22]. Increased urinary bladder stones, hyperplasia and transitional-cell carcinoma were observed in rats received 4500 mg/kg body weight/day of melamine for 103 week [23]. In vitro studies using neuronal cells indicated melamine can induce apoptosis [24], hyperpolarization and spontaneous firing [25] and also induce deficit of learning and memory in rats [26]. These studies were all carried out on adult animals or cell models. Either toxicology studies or kinetic studies using pregnant animals are still lacking. By administrating 800 mg/kg body weight/day from gestational day 6 to 20, maternal rat kidneys increased in weight and incidences of renal tubular necrosis, crystals, dilation and recruitment of inflammatory cells. However, no histological abnormality was observed in fetal organs, except less calcification of fetal skeleton [27]. The link between distribution and toxicity of melamine in fetal organs requires further detailed studies. In the present study, the significant levels of melamine in prenatal kidney during late pregnancy and in postnatal
C.Y. Chu et al. / Reproductive Toxicology 35 (2013) 40–47
kidney during early postnatal period suggested the significant distribution of melamine in the developing kidneys. It may imply the high susceptibility of melamine toxicity observed in infants after exposure to the tainted infant formulae in early age [28]. 5. Conclusions We have characterized the change in distribution of the melamine in different stages of pregnancy and during the development of the prenatal and postnatal organs. During the early pregnancy, the distribution of the melamine after a single oral intake was less, but increased during the course of pregnancy. Toward the end of the pregnancy, the developing kidney was shown to have the similar distribution as the other organs studied. This risk of melamine toxicity to kidney may increase after birth as demonstrated by the tremendous increase in the distribution of melamine observed in the 2-week old rats. Since the development of rat fetus is less mature compared to the human fetus, fetal kidney of rat is comparable to that of human in the 2nd and the 3rd trimesters. As a consequence, the toxicity of melamine may have already posted to human fetuses in late pregnancy. Acknowledgement This study was supported by a funding on Studies Related to Melamine Incident from Food and Health Bureau, Hong Kong Special Administration Region (MI-BS-06). References [1] Dakin HD, Dudley HW. Some limitation of the Kjeldahl method. Journal of Biological Chemistry 1914;12:275–80. [2] Brown CA, Jeong KS, Poppenga RH, Puschner B, Miller DM, Ellis AE, et al. Outbreak of renal failure associated with melamine and cyanuric acid in dogs and cats in 2004 and 2007. Journal of Veterinary Diagnostic Investigation 2007;19:525–31. [3] Chen JS. A worldwide food safety concern in 2008—melamine-contaminated infant formula in China caused urinary tract stone in 290,000 children in China. Chinese Medical Journal 2009;122:243–4. [4] Gossner CME, Schlundt J, Embarek PB, Hird S, Lo-Fo-Wong D, Beltran JJO, et al. The melamine incident: implications for international food and feed safety. Environmental Health Perspectives 2009;117:1803–8. [5] Baynes RE, Smith G, Mason SE, Barrett E, Barlow BM, Riviere JE. Pharmacokinetics of melamine in pigs following intravenous administration. Food and Chemical Toxicology 2008;46:1196–200. [6] Panesar NS, Chan KW, Lo WS, Leung VH. Ho CS Co-contamination, but not mammalian cell conversion of melamine to cyanuric acid the likely cause of melamine-cyanurate nephrolithiasis. Clinica Chimica Acta 2010;411: 1830–1. [7] Wu YT, Huang CM, Lin CC, Ho WA, Lin LC, Chiu TF, et al. Determination of melamine in rat plasma, liver, kidney, spleen, bladder and brain by liquid chromatography–tandem mass spectrometry. Journal of Chromatography A 2009;1216:7595–601.
47
[8] Chu CY, Chu KO, Chan JYW, Liu XZ, Ho CS, Wong CK, et al. Distribution of melamine in rat fetuses and neonates. Toxicology Letters 2010;199:398–402. [9] FDA. Interim safety and risk assessment of melamine and its analogues in food for humans [a]. http://www.fda.gov/Food/FoodSafety/ FoodContaminantsAdulteration/ChemicalContaminants/Melamine/ ucm164522.htm; 2008 [accessed 28.05.12]. [10] FDA. US Food and Drug Administration 2011 Dose Calculator. http://www.accessdata.fda.gov/scripts/cder/onctools/animalquery.cfm; [accessed 28.05.12]. [11] Chan JYW, Lau CM, Ting TL, Mak TCW, Chan MHM, Lam CWK, et al. Gestational and lactational transfer of melamine following gavage administration of a single dose to rats. Food and Chemical Toxicology 2011;49:1544–8. [12] CFS. Centre for Food Safety. Frequently asked questions. http://www. cfs.gov.hk/english/whatsnew/whatsnew fstr/whatsnew fstr Test dairy product FAQ.html; 2008 [accessed 28.05.12]. [13] Atherton JC, Pirie SC. The effect of pregnancy on glomerular filtration rate and salt and water reabsorption in the rat. Journal of Physiology 1981;319:153–64. [14] Zhang Q, Yang G, Li J, Li W, Zhang B, Zhu W. Melamine induces sperm DNA damage and abnormality, but not genetic toxicity. Regulatory Toxicology and Pharmacology 2011;60:144–50. [15] Varoe JS. Clinical biochemistry: techniques and instrumentation: a practical course. Singapore: World Scientific Publishing Co Ptc Ltd; 2001. pp. P5–9. [16] Sun Q, Shen Y, Sun N, Zhang GJ, Chen Z, Fan JF, et al. Diagnosis, treatment and follow-up of 25 patients with melamine-induced kidney stones complicated by acute obstructive renal failure in Beijing Children’s Hospital. European Journal of Pediatrics 2010;169:483–9. [17] Ding J. Childhood urinary stones induced by melamine-tainted formula: how much we know, how much we don’t know. Kidney International 2009;75:780–2. [18] Savin VJ, Beason-Griffin C, Richardson WP. Ultrafiltration coefficient of isolated glomeruli of rats aged 4 days to maturation. Kidney International 1985;28:926–31. [19] Chen WC, Wu SY, Liu HP, Chang CH, Chen HY, Chen HY, et al. Identification of melamine/cyanuric acid-containing nephrolithiasis by infrared spectroscopy. Journal of Clinical Laboratory Analysis 2010;24:92–9. [20] Puschner B, Poppenga RH, Lowenstine LJ, Filigenzi MS, Pesavento PA. Assessment of melamine and cyanuric acid toxicity in cats. Journal of Veterinary Diagnostic Investigation 2007;19:616–24. [21] Chen KC, Liao CW, Cheng FP, Chou CC, Chang SC, Wu JH, et al. Evaluation of subchronic toxicity of pet food contaminated with melamine and cyanuric acid in rats. Toxicologic Pathology 2009;37:959–68. [22] Hard GC, Flake GP, Sills RC. Re-evaluation of kidney histopathology from 13week toxicity and two-year carcinogenicity studies of melamine in the F344 rat: morphologic evidence of retrograde nephropathy. Veterinary Pathology 2009;46:1248–57. [23] Melnick RL, Boorman GA, Haseman JK, Montali RJ, Huff J. Urolithiasis and bladder carcinogenicity of melamine in rodents. Toxicology and Applied Pharmacology 1984;72:292–303. [24] Han YG, Liu SC, Zhang T, Yang Z. Induction of apoptosis by melamine in differentiated PC12 cells. Cellular and Molecular Neurobiology 2011;31: 65–71. [25] Yang J, An L, Yao Y, Yang Z, Zhang T. Melamine impairs spatial cognition and hippocampal synaptic plasticity by presynaptic inhibition of glutamatergic transmission in infant rats. Toxicology 2011;289:167–74. [26] An L, Li Z, Yang Z, Zhang T. Cognitive deficits induced by melamine in rats. Toxicology Letters 2011;206:276–80. [27] Kim SH, Lee IC, Lim JH, Shin IS, Moon C, Kim SH, et al. Effect of melamine on pregnant dams and embryo-fetal development in rats. Journal of Applied Toxicology 2011, http://dx.doi.org/10.1002/jat.1703. [28] Zhang L, Wu LL, Wang YP, Liu AM, Zou CC, Zhao ZY. Melamine-contaminated milk products induced urinary tract calculi in children. World Journal of Pediatrics 2009;5:31–5.