Diethylhexylphthalate Extracted by Typical Newborn Lipid Emulsions From Polyvinylchloride Infusion Systems Causes Significant Changes in Histology of Rabbit Liver  
From the * Clinic of Pediatric Surgery, University
of Marburg, Germany; Correspondence: P. D. Steffan Loff, MD, Clinic of Pediatric Surgery, Baldingerstraße, 35032 Marburg, Germany. Electronic mail may be sent to steffan.loff{at}web.de. Background: Looking for a candidate substance inducing hepatobiliary dysfunction under parenteral nutrition (PN) in newborns, we recently discovered that newborn infusions extract large amounts of the plasticizer diethylhexylphthalate (DEHP) from commonly used polyvinylchloride (PVC) infusion lines. This plasticizer is well known to be genotoxic and teratogenic in animals and to cause changes in various organs and enzyme systems even in humans. The aim of this study was to examine the effect of DEHP, extracted in the same way and in the same amount as in newborns, on livers of young rabbits. Methods: Prepubertal rabbits received lipid emulsion through central IV lines continuously for 3 weeks either via PVC or polyethylene (PE) infusion systems. Livers were examined after 1 and 3 weeks by light and electron microscopy. Results: By light microscopy, hydropic degeneration, single-cell necrosis, fibrosis, and bile duct proliferation were observed more in the PVC group. Electron microscopy revealed multiple nuclear changes, clusters and atypical forms of peroxisomes, proliferation of smooth endoplasmic reticulum, increased deposition of lipofuscin, and a mild perisinusoidal fibrosis only in the PVC group. These changes, which are generally regarded as reaction upon a toxic stimulus, could be exclusively attributed to DEHP. Conclusions: This investigation proved that DEHP produces toxin-like changes in livers of young rabbits in the same dose, duration, and method of administration as in newborn infants. For this reason, it is likely that DEHP is the substance that causes hepatobiliary dysfunction in newborns under PN. Possible modes of action of DEHP are proposed. Parenteral nutrition (PN) induces hepatobiliary dysfunction in man and especially in preterm infants after 3–4 weeks of PN only.1–3 Cause and therapy of this complication are still unsolved.4 In most cases, the disease is self-limiting when PN is completely stopped. But when PN must continue due to a functional or anatomical short bowel syndrome, severe liver damage follows and frequently the children die from complications of liver fibrosis.5 For this reason, the problem has a considerable clinical impact. Like other investigators, we were rapidly convinced that the liver damage is the effect of a toxic agent associated with PN.6,3 Many substances have been accused, among which were amino acids, especially methionine,4,6 toxic photodegradation products of PN,7 tryptophan,8 aluminum contaminations of the infusate,9 lithocholic acid produced under the conditions of enteral starvation,10,11 bacterial toxins due to bacterial overgrowth and bacterial translocation in the resting gut,12 and, recently, phytosterols, which have been proven to contaminate lipid emulsions frequently.13 Up to now, none of these possible causes have been convincing. Interestingly, methionine is toxic to the rat liver, but it is more toxic in combination with PN,14 and the same is true for aluminum.9 Bacterial translocation and toxins are relevant, but histologic typical hepatic lesions from PN differ from those of bacterial toxins.15,16 In an experimental study with weanling rats, Sokol et al17 detected that PN produces hepatic oxidative stress and glutathione depletion as soon as 5 days after initiation of PN. They found associated lipid peroxidation and hepatic injury but were not able to identify the source of oxidative stress. Consequently, our group began to look for a substance that fulfills the following demands:
We started thinking about plasticizer, extracted from polyvinylchloride (PVC) infusion lines, as a possible candidate substance and detected that PVC infusion lines, with which PN usually is administered worldwide, leach large amounts of the plasticizer diethylhexylphthalate (DEHP).18,19 The absolute amount extracted by typical infusions for newborns and for older children receiving home PN was measured to be comparably high.18 Thus, the dose:body weight ratio is much higher in newborns, indicating a higher threat to the latter. PN with PVC lines has invariably been correlated with the exposure of DEHP since the beginning of PN. DEHP has been proven to be carcinogenic20–24 and teratogenic25,26 and to cause damage in kidney,27 heart,28 liver,29 testis,30 and lungs31 of rodents and subprimates, but the liver was always regarded as the main target organ. Moreover, DEHP is suspected to affect kidney,27 heart,32 liver,33 lungs,34 and testis35 in man. Therefore, the theoretical demands concerning the accused substance were fulfilled by DEHP perfectly. The extrapolation of results from rat studies to man are frequently questioned because the metabolism of rats and man differs considerably,36 the doses of DEHP in man are usually dimensions lower than in animal experiments,24 and the access for the highest doses in man is parenteral and in experimental animals through gavage. For these reasons, the load of DEHP extracted by typical newborn lipid infusions from PVC infusion lines was measured. We wondered whether this amount of DEHP would change liver histology in prepubertal rabbits when administered using the same IV access and the same time period as in infants. Rabbits were used because this model was successfully established in our department for long-term PN. Because it is well known that young livers in general are more prone to toxic changes and because it was our intention to imitate in the experiments the situation of an infant as well as possible, we choose young rabbits for the experiments.
The animal experiments were carried out in accordance with German law and approved by the commission for laboratory animal protection, Karlsruhe. Prepubertal rabbits (about 1–1.5 kg) obtained from Harlan (Harlan Winkelmann GmbH, Borchen, Germany) were housed in individual cages with a 12-hour dark-light cycle, and controlled temperature (21°C) and moisture (50% relative saturation). Central venous access was established after 1 week of acclimatization. For this reason, animals were anaesthetized with xylazine and ketamine. The right lateral neck and interscapular region was carefully shaved and the animal fixed in a supine position, with the head turned left. Ointment was administered to protect the eyes from drying. A longitudinal skin incision at the right lateral neck exposed the V. maxillaris. A 20 gauge, 12-cm polyurethane catheter (Arrow International, Reading, PA) cut to the appropriate length was introduced and pushed forward into the brachiocephalic vein. The end of the catheter was exteriorized between the ears, thus producing a long subcutaneous tunnel to prevent the insertion area from infection. The exact procedure is described in detail by Zovko et al.37 After 1 week of healing and recovery from the operation, infusions started. Fifteen prepubertal rabbits received daily 24 mL of lipid infusions continuously through PVC infusion systems for 3 weeks. This duration was chosen because long-term histologic liver changes in infants are usually seen after 3 weeks. One and 2 mg of DEHP were extracted by 24 mL of lipid infusion from the PVC line. This was measured by mass absorption–gas chromatograph and proved a daily load of DEHP of the rabbits in this group of 1 mg/kg. Ten prepubertal rabbits received the same lipid emulsions for the same time through polyethylene (PE) infusion systems. Except for this difference, the procedures in both groups were identical. Nine rabbits with no infusions or manipulation served as controls. After 1 week, liver specimens were obtained by open surgery as described,37 and after 3 weeks the animals were killed and the livers obtained. All liver specimens were divided into 2 pieces. One portion was fixated in buffered 3% formalin solution, embedded, cut, and stained with hematoxylin-eosine (HE). Then a pathologist examined the slides with light microscopy. A histologic scoring system was used as described for rabbit livers by Curran et al38: The histologic findings were scored on a scale of 0–3+. For balloon degeneration, 1+ indicates that ballooned cells extend <25% of the distance from the terminal hepatic vein to the portal triad, 2+ indicates that they extend 25%–50% of the distance, and 3+ indicates that they extend >50% of the distance. For portal inflammation, 1+ indicates that inflammatory cells are present but fill <25% of the area of the portal triad, 2+ indicates that they fill 25%–50% of the area, and 3+ indicates that they fill 50%–100% of the area. For portal fibrosis, 1+ indicates fibrosis limited to the triad area, 2+ indicates fibrosis extending beyond the triad, and 3+ indicates fibrosis with portal-to-portal bridging. For bile duct proliferation, the other portion was immediately fixed and processed for electron microscopical examination. The main steps of the procession of liver tissue samples for electron microscopy were as follows: the tissue samples were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4). After fixation, the samples were washed with cacodylate buffer (4°C, 30 minutes) and then incubated in 1% reduced osmium tetroxide for 90 minutes. This was followed by a series of washing steps, first with cacodylate buffer followed by maleate buffer (0.05 M, pH 5.2). Thereafter, the samples were stained en bloc overnight in 1% uranyl acetate in maleate buffer. The incubation was followed by washing with maleate buffer. The samples were then dehydrated over an increasing alcohol gradient, starting with ethanol 75% (4°C, 30 minutes for each step). The dehydration was finished with 100% ethanol (20°C, 30 minutes). The samples were embedded in Spurr embedding medium according to Spurr.39 Polymerization of the samples in embedding medium was conducted into an embedding oven at 60°C over a period of 3 days (Agar Scientific, Essex, England). Ultrathin sections (50–80 nm) were cut from the embedded samples using a diamond knife (Diatome AG, Biel, Schweiz) mounted on a Leica Ultracut R Ultramicrotome (Leica, Bensheim, Germany). The sections were collected on copper grids and were counter-stained with alkaline lead citrate. Ultrastructural examination of the section was performed using a Zeiss EM 9S electron microscope (Carl Zeiss, Jena, Germany). All chemicals used were purchased from Serva (Serva, Heidelberg Germany).
These studies were carried out by a biologist specializing in electron microscopy.
In light microscopy, the livers of the control animals without any treatment had a portal fibrosis of a minor degree (scoring 0.72) at autopsy (Figure 1). Half of the animals showed a few scattered mononuclear cells in the portal area. Single-cell necrosis and hydropic or fatty degeneration were almost absent. Fibrosis and bile duct proliferation were less pronounced in the control group (0.72 and 0.22; Figure 1) and the PE group (1.5 and 0.4; Figure 2) compared with the PVC group under the effect of DEHP (1.76 and 0.56; Figure 3). The PVC group showed remarkably more single-cell necrosis and foci of necrosis (0.9) than the PE group (0.25) and control group (0.05). Degenerative changes were low in the PVC and PE group but even more pronounced in the PVC group. For more details see Table I.
Via electron microscopically, the following changes were found. In the control group (Figure 4), hepatocytes showed a round nucleus. The rough endoplasmic reticulum (rER) exhibited a slight tendency to dilatation. Mitochondria were homogenous except for a few atypic forms. Peroxisomes and the Golgi apparatus were normal, as were the canaliculi. Lysosomes appeared sporadic. Glycogen content of the cells was considered to be moderate. The sinusoids had typical endothelial cells and normal Kupffer cells. The livers of the PE group (Figure 5) showed a mildly disturbed cell compartmentation. Changes of the nucleus were unremarkable and much less pronounced than in the PVC group. Peroxisomes were slightly increased, without clusters or atypic forms. The rER had a clear tendency of vesiculation. There was only weak proliferation of the smooth endoplasmic reticulum (sER). The canaliculi had a stronger atrophy of microvilli, and myelin bodies were more prominent than in the PVC group. The sinusoids were inconspicuous. In the PVC group (Figure 6), the livers had a clearly disturbed cell compartmentation. The nucleus showed frequently heterochromatin condensation in the periphery, increased number of nucleoli with peripheral displacement, and striking structure. The peroxisomes were increased, showing clusters, atypical forms, and marked paracrystalline structures. There was a clear proliferation of sER, with a local association to the peroxisomes. Lysosomal structures were increased, especially lipolysosomes and depositions of lipofuscin. The glycogen content was reduced. Within the sinusoids, slight changes of the Kupffer cells and a mild perisinusoidal fibrosis were found (Table II).
We suspected DEHP, extracted from PVC infusion lines, to be involved in PN-induced hepatobiliary dysfunction of neonates. Our prior investigation of the leachability revealed that large amounts of DEHP are extracted by lipid emulsions from PVC infusion lines under conditions typical for premature newborns.18,19 In the experiments presented here, young rabbits were exposed to DEHP (see Materials and Methods) comparable in dose, duration, and way of access to those in neonates. The only difference between the 2 experimental groups was the use of PE infusion systems in 1 group and PVC infusion systems in the other, implicating a dose of 1 mg/kg/d of DEHP in the PVC group. Differences in liver histology could be attributed exclusively to this fact. We found obvious differences of the liver specimens between both groups in light and electron microscopy. The changes in light microscopy indicated a toxic effect in the PVC group with increased cell necrosis, fibrosis,40 and bile duct proliferation after 3 weeks of application. These results were supported by characteristic differences between both experimental groups in electron microscopy. Here, the PVC group showed changes of the nucleuslike heterochromatin condensation in the periphery and proliferation of sER, which are typical electron microscopical signs for toxic damage.41 Increased number, clusters, and atypical forms of peroxisomes, as well as augmented lipofuscin pigment, is a sign of increased oxidative stress.41 The detection of perisinusoidal fibrosis was in accordance with the increased fibrosis seen in light microscopy.
In conclusion, there can be no doubt that the animals of the PVC group experienced a toxic effect and an enhanced oxidative stress, whereas the animals from the PE group did only to a minor degree. These effects in the PVC group must be attributed to the extraction of DEHP from the infusion lines. Still it remains to be elucidated why the PE group showed mild but visible changes. It is unclear whether this was a general reaction of the livers to lipid emulsion or a minor toxic effect due to the DEHP immanent in the lipid emulsion before perfusion of the PVC lines. It is well known that DEHP alters the activity of multiple liver enzymes, especially those of phase I and II of biotransformation of xenobiotics. Two possible means of action are shown in Figure 7, modified from a scheme of the Oxford Textbook of Pathology.41 Both acetaminophen and menadione are commonly used in infants. When the oxidoreductase is blocked in the metabolism of menadione, alternative pathways produce a highly liver-toxic intermediate. In the case of dicumarol, the enhanced toxicity of menadione on the liver is proved. DEHP is proved to inhibit the oxidoreductase as well in rat testis by 45%.42
In the metabolism of acetaminophen, a liver-toxic intermediate is generated when the common pathway is saturated or the toxic alternative pathway is stimulated. A stimulation of the cytochrome P-450 group by DEHP in animal livers has been shown by several investigators.33,43–46 The toxic effects on rabbit livers in the PVC group of our experiments, as well as toxic effects on the liver of infants receiving long-term PN with PVC systems, can be easily explained: We suggest that the capacity of the liver cells to detoxify endogenous or exogenous toxins is impaired by DEHP through inhibition of detoxifying enzymes or stimulation of toxifying metabolic pathways, thus generating toxic effects and oxidative stress visible in light and electron microscopy. This new idea in understanding PN-induced hepatobiliary dysfunction would explain many clinical observations of this disease and supposed contradictions of different experimental investigations. One next step would be to prove an effect of DEHP doses on human liver tissue. For this reason, we are currently investigating human liver cell cultures exposed to DEHP, which is extracted by lipid emulsions from PVC infusion lines. Received for publication July 13, 2006. Accepted for publication October 12, 2006.
Journal of Parenteral and Enteral Nutrition, Vol. 31, No. 3,
188-193 (2007)
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MATERIALS AND METHODS
RESULTS
