Main

Neonatal jaundice is common during the transitional period and is exacerbated by factors that increase bilirubin production rates, such as hemolytic disease(1). Currently, the more common methods of treatment for exaggerated hyperbilirubinemia include facilitation of alternate pathways of bilirubin excretion through phototherapy and removal of plasma bilirubin via exchange transfusion(2). However, reducing bilirubin production is an attractive alternative chemopreventive strategy for those infants at risk for severe hyperbilirubinemia due to increased production of the pigment.

Microsomal HO (EC 1.14.99.3) is the rate-limiting enzyme in the catabolism of heme to bilirubin(3) and exists in two isoforms, HO-1 and HO-2(4, 5). The regulation of the activity of these isoforms, and thus heme turnover, is both clinically and physiologically important because of its critical role in bilirubin production and maintenance of heme and hemoprotein homeostasis. The most well studied metalloporphyrins, SnPP and SnMP, have been shown to inhibit total HO activity in vivo(6, 7) and, as a result, to suppress bilirubin production(8) and reduce serum bilirubin levels(6, 7) in animal models. In clinical trials, SnPP and more recently SnMP were reported to significantly reduce the degree of hyperbilirubinemia in newborn infants(9, 10) and Crigler-Najjar patients(11, 12). However, despite their clinical utility, chronic or higher dose administration of SnPP or SnMP has also been associated with photosensitization after exposure to broad spectrum light(9, 10, 13) and disruption of iron homeostasis(14). In animal models, the administration of SnPP at high doses has also been associated with1) phototoxicity(15, 16); 2) brain metalloporphyrin deposition, decreased HO activity, and modestly altered glutathione levels(17–19); 3) decreased tissue HO-2 protein levels(19, 20);4) altered activities of P450-dependent enzymes(21, 22); and 5) increased HO-1 mRNA and protein levels(20, 23).

Other metalloporphyrins, such as ZnPP, zinc mesoporphyrin, zinc deuteroporphyrin bis-glycol, chromium protoporphyrin, and chromium mesoporphyrin, are also effective inhibitors of HO activity(24–28) and suppress bilirubin production(24, 25, 29, 30) and serum bilirubin levels(28, 29, 31). Although some of these metalloporphyrins may also exhibit side effects when administered at high doses in animal models(20, 25), pharmacologic and toxicologic information is limited. Of these compounds, ZnPP is the most well studied alternative to the tin porphyrins, is minimally photoreactive in vitro(32), and not phototoxic in vivo(16). Regardless, the safety of ZnPP remains to be fully established, as in vitro studies have reported cell culture growth inhibition(33) and possible interference with signal transduction(34). However, ZnPP reportedly does not alter brain glutathione concentrations(35), HO-2 protein levels(19, 20), or hepatic P450-dependent enzyme activities(26, 31). Furthermore, as a required trace element, free zinc should be metabolized with minimal toxicity, if the metal disassociated from the macrocycle in vivo. Therefore, we believe ZnPP has potential as an alternative chemotherapeutic agent for the treatment of severe hyperbilirubinemia and warrants further investigation with regard to its duration of action and tissue distribution.

Current pharmacologic information on ZnPP is limited, although its biliary excretion(31) and poor enteral absorption have been previously documented(36). Previous studies of other metalloporphyrins have shown that liver, spleen, kidney, and brain(18) are potential deposition sites, and that the duration of action ranges from 2 to 11 d, depending upon animal model, route of administration, dose, and metalloporphyrin(18, 31). However, ZnPP also induces HO-1 gene expression in vivo(20), and it is possible that this may blunt its apparent ability to inhibit HO activity, as speculated for other metalloporphyrins(12, 20). As a result, duration of action measurements would be strengthened by confirmation that HO inhibition was not influenced by a simultaneous change in HO-1 expression. Therefore, the aim of the present study was to investigate the duration of HO inhibition, suppression of serum bilirubin levels, HO-1 gene expression, and tissue distribution of ZnPP after i.p. administration at 40 μmol/kg B.W. to neonatal rats. Toward this goal, we determined and compared changes in total HO activity, plasma bilirubin levels, the HO-1 mRNA and protein levels, and ZnPP concentrations in plasma, liver, spleen, kidney, lung, brain, and erythrocytes for up to 21 d after dosing.

METHODS

Chemicals and materials. AmpliTaq DNA polymerase was obtained from Perkin-Elmer Corp. (Norwalk, CT); all other enzymes were purchased from Life Technologies, Inc. (Gaithersburg, MD). Chemicals were obtained from Sigma Chemical Co. (St. Louis, MO). ZnPP (formula weight 625.9) was obtained from Porphyrin Products, Inc. (Logan, UT). Stock (8.0 mM) ZnPP solutions were prepared fresh daily as 25.1 mg of ZnPP dissolved in 0.25 mL of 10% (vol/vol) ethanolamine, diluted in 0.1 M sodium phosphate buffer, titrated to pH 8.0 with 1 N HCl, and brought to a final volume of 5 mL. Diluent (carrier) was similarly prepared without ZnPP. Solutions were prepared in dim light, stored in the dark, and used within a few hours of preparation.

Animals. Litters of Wistar rats (12 pups per litter) were housed singly, and all animals were allowed free access to food and water before and after dosing. Litters were housed in a temperature-controlled room(25 ± 4°C) on a 12-h light cycle. All animal care was in accordance with National Institutes of Health guidelines and under institutional animal care and use committee approval.

Experimental design. Before dosing, neonatal rats were weighed and randomly assigned to either control or ZnPP-treated groups. All subsequent experimental procedures were performed in dim light. Rats were 24-36 h of age at dosing. Animals assigned to the ZnPP treatment group were injected intraperitoneally with ZnPP at 40 nmol/g B.W.; controls were injected with an equivalent volume (5 μL/g B.W.) diluent solution. The average B.W. at time of dosing was 7.4 ±.82 g for ZnPP-treated neonates and 7.4 ± 0.85 g for control neonates; therefore, the average dose was 296 nmol of ZnPP. At selected time points between 12 h and 21 d after dosing, neonatal rats were anesthetized with CO2 and killed by decapitation. To determine plasma and red blood cell ZnPP content, heparinized blood samples were obtained by pooling from every three animals. To determine serum bilirubin levels, blood samples were obtained by pooling from every two animals. Blood samples were centrifuged, and the red blood cells were separated from the plasma or serum. For tissue ZnPP analysis and HO activity measurements, liver, brain, and lung samples were each obtained by pooling from every two animals; kidney and spleen samples were each obtained by pooling from every three animals. Tissues were homogenized in 4 volumes of 0.1 M sodium potassium phosphate buffer, pH 7.4. For tissue HO-1 mRNA determinations, liver samples were obtained by pooling from every two animals; spleen samples were obtained by pooling from every three animals. Samples were immediately homogenized in 4 M guanidinium thiocyanate(37), quickfrozen in liquid N2, and stored at -80°C. Because sample values were determined using organs or blood pooled from either two or three animals, the n values given underrepresent the number of animals used by one-half or two-thirds, respectively.

Tissue HO activity. Total HO activity was determined as described previously in 13,000 × g supernatants prepared from tissue homogenates based on a gas chromatographic method(38). The final reaction mixture contained 50 μM methemalbumin (2.4 μM albumin), 1.5 mM NADPH, and 13,000 ×g supernatant as a source of HO. Activity is expressed as nmol of CO/h/mg of protein. The limit of detection of this assay is 1 pmol of CO per vial. Supernatants were analyzed for protein content according to the method of Lowry et al.(39), using BSA as a standard.

Serum bilirubin determinations. Aliquots (25 μL) of serum were analyzed for total bilirubin concentration photometrically at 450 nm using the UB analyzer (model UA-1, Arrows Co., Ltd., Osaka, Japan), according to the method of Nakamura and Lee(40).

Fluorescence analysis of tissue ZnPP. Aliquots (50 μL) of plasma and tissue homogenates were vigorously extracted with 0.25 mL of a mixture of acetone:0.5 M ammonium acetate, pH 4.5 (80:20, vol/vol). After centrifugation at 13,000 × g, the supernatant (extract A) was decanted into a separate tube. The pellet was reextracted with 0.25 mL of a mixture of methanol:ethanolamine (99:1, vol/vol), centrifuged, and the supernatant (extract B) decanted. Each extract was then diluted in methanol:0.5 M ammonium acetate, pH 4.5 (80:20, vol/vol) and the fluorescence(relative intensity, RI) was determined using a Hitachi F4010 spectrofluorometer (excitation = 416 nm, emission = 588 nm). ZnPP standards (8μM in 0.1 M sodium phosphate buffer, pH 8.0) were prepared immediately before use and diluted in methanol:0.5 M ammonium acetate, pH 4.5 (80:20, vol/vol), and fluorescence was measured as described. The limit of detection of this method was determined to be approximately 0.2 nM, which is approximately equivalent to 0.25 pmol of ZnPP in the 50-μL sample aliquots. Plasma and tissue extract emission and derivative spectra were also recorded to confirm the presence of ZnPP(41).

Calculation of tissue ZnPP content. The fluorescence yield of extract A and B collected from ZnPP-treated animal tissues was corrected for background fluorescence (determined from diluent-treated neonatal rat tissue extract A and B). The ZnPP concentrations of extract A and B were then calculated using ZnPP standard curve equations. The ZnPP tissue concentrations(in nmol/g) were determined according to Equation 1: [extract A (μM ZnPP) + extract B (μM ZnPP)] × dilution factor × extract volume÷ extraction efficiency ÷ sample volume × homogenate volume ÷ wet tissue weight = nmol/g ZnPP. Plasma ZnPP concentrations(μM) were determined similarly, but without correction for homogenate volume and tissue wet weight.

ZnPP extraction efficiencies from plasma, erythrocytes, and tissues were determined in a series (n ≥ 4) of separate experiments by spiking fresh rat plasma or tissue homogenates with varying amounts of ZnPP standard, ranging from 0.01 to 9.25 nmol, to mimic experimental levels observed in the corresponding tissue. Mixtures were extracted as described, and the nanomoles of ZnPP recovered were calculated. These extraction efficiencies, as used in subsequent calculations, were as follows: plasma, 103%; liver, 64.5%; spleen, 59.4%; kidney, 59.5%; brain, 68.5%; lung, 52.2%; and erythrocytes, 60.4%.

Because the increasing tissue mass in the growing neonatal rats may underestimate the apparent ZnPP tissue concentration (in nmol/g), the total ZnPP content of each tissue (in nmol) was also calculated according to Equation 2: [extract A (μM ZnPP) + extract B (μM ZnPP)] × dilution factor × extract volume ÷ extraction efficiency ÷ sample volume × homogenate volume = nmol ZnPP. The total plasma and erythrocyte ZnPP content (in nmol) was determined similarly, substituting the estimated plasma or red blood cell volume (assuming normal rat blood volumes of 58 mL/kg and hematocrit values of 46 mL/100 mL) for homogenate volume.

The percent of the administered ZnPP dose recovered in tissue, plasma, and erythrocytes was calculated by dividing the nanomoles of ZnPP recovered(defined in Equation 2) by the administered dose (in nmol).

Plasmid and probe preparation. The plasmid pBKRHO1 was constructed in pBluescript II SK- using a rat HO-1 cDNA fragment prepared by reverse transcription-polymerase chain reaction that corresponded to HO-1 nucleotides -33 to +833 as reported by Shibahara et al.(42). Poly(A+) RNA was isolated from rat spleen(Poly(A) Quik mRNA isolation kit, Stratagene, La Jolla, CA), and used for first strand cDNA synthesis (Superscript II kit; Life Technologies, Inc.) using an oligo(dT)15 primer (Promega, Madison, WI). HO-1-specific oligonucleotides were obtained from Eppendorf (Madison, WI); the 5′-sense oligonucleotide (5′-GCAAGCTTAGCGGAGCCAGCCTGAA-3′) corresponded to residues -33 to -16 and contained six additional nucleotides coding for a HindIII restriction site; the 3′-antisense oligonucleotide (5′-GGCTCGAGGAAACTGAGTGTGAGGA-3′) corresponded to residues 814-833 and contained eight additional nucleotides coding for anXho I restriction site. After 2 × 30 cycles of amplification(GeneAmp polymerase chain reaction reagent kit, Perkin-Elmer Corp.), the polymerase chain reaction product was phenol-chloroform-extracted and ethanol-precipitated. The resuspended cDNA was digested with HindIII and XhoI, gel purified, and cloned into pBluescript II SK-. The resulting plasmid (pBKRHO1) was used to transform Escherichia coli DH 5∂ (Life Technologies, Inc.). Transformants were screened by restriction digestion, and the nucleotide sequence of the insert (RHO-1 cDNA) was confirmed by the dideoxynucleotide chain termination method (PAN Facility, Beckman Center, Stanford University). The housekeeping gene GAPD cDNA(43) (American Type Culture Collection; Rockville, MD) was prepared as an EcoRI digest of HHCPF19 plasmid by standard methods(44). Labeled probes were prepared by the random primer method(45) using [32P]dATP.

Northern hybridization. RNA was isolated by the guanidinium thiocyanate:phenol extraction method of Chomczynski and Sacchi(37) and quantitated spectrophotometrically at 260 nm. RNA (20 μg) was denatured, electrophoresed on a 1.2% agarose gel containing 1.1 M formaldehyde, transferred to a positively charged nylon membrane (Hybond N+; Amersham Corp., Arlington Heights, IL) and immobilized by UV irradiation (UV-Stratalinker; Stratagene, La Jolla, CA). Membranes were prehybridized for 4-6 h at 42°C in buffer containing 50% formamide, 5× SSPE, 5 × Denhardt's solution, 1% SDS, 10% dextran sulfate, and 50 μg/mL denatured salmon sperm DNA, and subsequently hybridized for 18-24 h at 42°C in buffer containing 50% formamide, 5 × SSPE, 5 × Denhardt's solution, 1% SDS, 10% dextran sulfate, and 32P-labeled probe(5-10 × 108 cpm/μg, 1-5 × 106 cpm/mL). Membranes were washed twice at 25°C for 5 min in 2 × SSPE, 0.1% SDS, and twice at 50°C for 20 min in 0.2 × SSPE, 0.1% SDS, dried briefly, and exposed to Kodak X-Omat film with an intensifying screen at -80°C. For reprobing, membranes were stripped according to the manufacturer's protocol using boiling 0.5% SDS. HO-1 and GAPD mRNA quantification was performed by densitometry (PDI, Santa Clara, CA), and statistical analysis was performed on the ratio of HO-1 to GAPD. Normalization to GAPD was selected on the basis of preliminary studies that indicated that ZnPP had no apparent effect on the expression of this housekeeping gene. We also determined that the addition of ZnPP directly to total RNA samples isolated from control animals did not affect Northern hybridization procedures or signal detection.

Western analysis. Aliquots (50 μg) of 13,000 ×g supernatants were electrophoresed on a 12% polyacrylamide gel according to the method of Laemmli(46). Proteins were transferred for 2 h to polyvinylidene difluoride membrane (Bio-Rad, Richmond, CA) with a Bio-Rad Mini Trans-Blot apparatus according to the method of Towbinet al.(47). Blots were blocked overnight at 25°C in 10% nonfat milk, washed briefly with Tris-buffered saline containing 0.1% Tween 20, incubated for 2 h with 1:300 dilution of rabbit anti-rat HO-1 IgG, and washed with Tris-buffered saline. Antigen-antibody complexes were visualized with the alkaline phosphatase chemiluminescent system according to the manufacturer's instructions (Bio-Rad). Microsomes prepared from cobalt chloride-treated rat liver were used as a positive control for identification of the HO-1 protein. Quantification was performed by densitometry (PDI, Santa Clara, CA).

Preparation of HO-1 antibodies. Antisera was prepared by Berkeley Antibodies, Inc. (Berkeley, CA). Polyclonal rabbit anti-rat HO-1 antibodies were raised against a 30-kD soluble HO-1 protein expressed inE. coli using rat liver cDNA(48) (gift of A. Wilks, University of California, San Francisco, CA). Antisera was precleared by incubating at 4°C with normal rat serum for 2 h, followed by 10%Staphylococcus aureus protein A-Sepharose CL-4B beads (Sigma Chemical Co.) for an additional 2 h. After centrifugation at 6,000 ×g for 5 min, the supernatant was recovered and used as a source of HO-1 antibody for Western analysis.

Statistics. Data are reported as the mean ± SEM. Statistical significance (p < 0.05) was determined by single factor analysis of variance and Fischer's protected least significant difference method of multiple comparisons (Statview 4.02, Abacus Concepts, Inc., Berkeley, CA).

RESULTS

Body and organ weights. ZnPP administration had no apparent adverse effect on the growth of neonatal rats which averaged 2.4 g/d in both groups during the 21-d experimental period. Furthermore, no significant differences in body or organ weights (liver, spleen, lung, kidney, and brain) were observed between controls and ZnPP-treated animals at any time point(data not shown).

Total HO Activity. As expected, control neonatal hepatic HO activity peaked between 1 and 4 d after dosing (≈2-5 d after birth). Activity in the liver was significantly inhibited in ZnPP-treated neonatal rats compared with controls between 1 and 4 d after dosing (Fig. 1). The degree of inhibition ranged from 27% on d 1 to a maximum of 51% on d 3. Although hepatic HO activity in ZnPP-treated neonates was inhibited by 26% on d 7 after dosing, this was no longer significant. By d 21 after dosing, liver HO activity had returned to control levels. In both control and ZnPP-treated neonatal rats, splenic HO activity gradually increased, rising from approximately 0.7 nmol/h/mg at 12 h after dosing (≈2 d after birth) to 2.1 nmol/h/mg at 21 d after dosing. These developmental changes in liver and spleen HO activity are consistent with those results reported by Maines and Kappas(49). In contrast to the liver, there were no remarkable or significant differences between ZnPP-treated and control neonatal rat splenic activities (data not shown).

Figure 1
figure 1

Total HO activity in 13,000 × g supernatants prepared from the liver of neonatal rats at selected time points between 0 and 21 d after i.p. administration of either 40 nmol/g B.W. ZnPP or 5 μL/g B.W. diluent. Neonatal rats were 24-36 h old at the time of dosing. For each time point n ≥ 6, where n represents the number of pooled samples analyzed; *p ≤ 0.05; ‡p ≤ 0.001.

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Serum bilirubin levels. In control neonates, the serum bilirubin concentration was greatest on d 1 after dosing (≈2 d after birth) and thereafter gradually declined. In ZnPP-treated neonatal rats, reductions in serum bilirubin after dosing ranged from 28% on d 1 to 23% on d 4, but were significantly different (p = 0.0003) only on d 1(Table 1).

Table 1 Serum bilirubin levels (μmol/L) in neonatal rats at selected time points after administration of ZnPP (40 nmol/g B.W.) or diluent (5 μl/g B.W.)
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Metalloporphyrin content. As shown in Figure 2A, extract emission spectra from the liver, spleen, kidney, lung, brain, and plasma corresponded to the ZnPP standard spectrum. All extracts exhibited peak emissions at 588 nm, which indicates to the presence of ZnPP rather than free protoporphyrin. In brain tissue samples, where low ZnPP levels often precluded quantitation, the presence of ZnPP was qualitatively confirmed through second derivative spectra, which records the change in slope of fluorescence relative intensity as a function of emission wavelength(41). As shown in Figure 2B, the trough of both the brain extract and ZnPP standard derivative spectra corresponded to 588 nm.

Figure 2
figure 2

(A) Fluorescence spectra of ZnPP standard solution (Z) and acetone:acetate extracts of liver (Li), spleen (Sp), kidney (Ki), and lung (Lu) homogenates and plasma (Pl) prepared from neonatal rats after i.p. administration of a 40-nmol/g B.W. dose. Excitation wavelength, 416 nm; peak emission wavelength, 488 nm. (B) Second derivative spectra of ZnPP standard solution (Z) and acetone:acetate extracts of brain homogenate (Br) prepared from neonatal rats after ZnPP dosing.

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The greatest concentration of ZnPP was observed in neonatal rat liver. The hepatic ZnPP concentration was 2-5-fold greater than that of the spleen and 5-10-fold greater than that of the kidney. ZnPP deposition in these tissues accounted for the majority of the recovered dose. As shown in Figure 3, the ZnPP concentration (nmol/g tissue) in these tissues was greatest between 1 and 4 d after dosing (≈2-5 d after birth), and then progressively decreased. However, concentration values,i.e. nanomoles/g tissue, may be misleading in growing neonatal animals where tissue mass is increasing. Therefore, we also examined the total nanomoles of ZnPP found in each tissue (Fig. 4). In the liver, the total nanomoles of ZnPP found followed a pattern similar to the changes in concentration. In contrast, the total nanomoles of ZnPP found in the spleen and kidney were still elevated at 14 and 21 d after dosing, respectively, suggesting retention of the metalloporphyrin during organ development. In plasma, the ZnPP concentration (μM) progressively declined (Fig. 5) to barely detectable levels after 7 d, whereas the total nanomoles of ZnPP found in plasma remained elevated for 2-3 d after dosing. This may be due to continued absorption from the intraperitoneal injection site, and is not surprising, because residual ZnPP was observed in the peritoneal cavity for at least 7 d after dosing. Specifically, we observed a dark purple pastelike material which adhered to the peritoneal wall and to organs within the peritoneal cavity, which was identified by fluorometric analysis as ZnPP. However, it was not possible to accurately quantitate the amount of material remaining.

Figure 3
figure 3

ZnPP concentrations (nmol/g) in liver, spleen, and kidney homogenates prepared from neonatal rats at selected time points between 0 and 21 d after i.p. administration of a 40 nmol/g B.W. dose. Neonatal rats were 24-36 h old at the time of dosing. For each time point, n ≥ 7 in each tissue, where n represents the number of pooled tissue samples analyzed.

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Figure 4
figure 4

Total ZnPP (nmol) recovered in the liver, spleen, and kidney of neonatal rats at selected time points between 0 and 21 d after i.p. administration of a 40 nmol/g B.W. dose. Neonatal rats were 24-36 h old at the time of dosing. For each time point, n ≥ 7 in each tissue, wheren represents the number of pooled tissue samples analyzed.

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Figure 5
figure 5

Plasma ZnPP concentration (μM) in neonatal rats at selected time points between 0 and 21 d after i.p. administration of a 40 nmol/g B.W. dose. For each time point n ≥ 7, where n represents the number of pooled tissue samples analyzed.

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Lung, brain, and erythrocytes were not significant ZnPP deposition sites in neonatal rats after i.p. dosing (Table 2). In the lung, the concentration of ZnPP was greatest at 2 and 3 d after dosing (1.8 ± 0.2 and 1.4 ± 0.3 nmol/g tissue, respectively), and then progressively decreased to less than 0.1 nmol/g by 14 d after dosing. A similar trend in the lung total nanomoles of ZnPP was also observed. The concentration of ZnPP in the brain was very low, averaging from 0.02 ± 0.02 to 0.05 ± 0.02 nmol/g tissue during the first 4 d after dosing. By d 7, the level of ZnPP in the brain had decreased further, and was detectable in only one out of the eight samples examined. In contrast to the plasma ZnPP level, the total nanomoles of ZnPP found in neonatal rat circulating erythrocytes remained measurable at 14 d (0.52 ± 0.10 nmol; 0.18 ± 0.03% of the administered dose), then decreased to 0.15 ± 0.05 nmol at 21 d after dosing.

Table 2 ZnPP tissue distribution in neonatal rats at selected time points after administration of a 40-nmol/g B.W. (i.p.) dose*
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Hepatic HO-1 mRNA levels. At 12 h after ZnPP dosing, the ratio of HO-1 to GAPD steady state mRNA levels (Fig. 6) was 2.5-fold greater (p < 0.001) than that of control animals(Table 3). This increase was not due to ZnPP-induced changes in GAPD mRNA levels, because these were not significantly different between control and treated neonates. By d 1 after dosing, HO-1 to GAPD mRNA ratios were not significantly different between the two groups. Furthermore, no significant differences were observed at later time points.

Figure 6
figure 6

Northern analysis of steady state hepatic HO-1 and GAPD mRNA levels in neonatal rats at 4, 8, 12, 24, and 48 h after i.p. administration of either 40 nmol/g B.W. ZnPP (Z) or 5 μL/g B.W. diluent (C). Equal amounts of total RNA (20 μg) were loaded in each lane.

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Table 3 HO-1/GAPD mRNA ratios in neonatal rats at selected time points after administration of ZnPP (40 nmol/g B.W.) or diluent(5 μl/g B.W.)
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Hepatic HO-1 protein levels. At 24 h after dosing, a modest but statistically significant (p < 0.025) increase in steady state HO-1 protein level was observed in ZnPP-treated neonatal rats (1.07 ± 0.10, n = 3) compared with controls (0.60 ± 0.10, n= 3). However, this apparent induction was short-lived, and no differences were observed at 48 h (0.841 ± 0.18, n = 3 controls; 0.98± 0.23, n = 3 ZnPP-treated) or 72 h after dosing (0.74± 0.11, n = 3 controls; 0.93 ± 0.08, n = 3 ZnPP-treated).

DISCUSSION

ZnPP has been shown to inhibit total HO activity in vivo(26, 31) and reduce bilirubin production rates(indexed through exogenous carbon monoxide excretion rates) in adult rats(50) and neonatal nonhuman primates(29, 31, 51). In these studies, which differ in dosing schedule and route of administration, inhibition of hepatic HO activity was detected as early as 12 h in adult rats and as late as 11 d in neonatal non-human primates. Suppression of serum bilirubin levels in neonatal non-human primates has been observed over a similar time period(31, 51). However, the time course of HO inhibition and pattern of ZnPP tissue deposition has not been extensively explored.

In the current study, the duration of action of ZnPP in neonatal rats was less than 7 d. The significant decreases in total hepatic HO activity between d 1 and 4 after dosing corresponded well with peak liver ZnPP concentrations. By comparison to SnPP, the most widely studied metalloporphyrin, this duration of action is greater than the 2 d of hepatic HO inhibition reported in neonatal rats exposed to SnPP antenatally (50 μmol/kg B.W. administered to the dam)(18). However, in adult rats, the duration of HO inhibition is similar to that observed after ZnMP dosing (15 μmol/kg B.W., i.v.)(27), but less than that reported for SnMP(1 μmol/kg B.W., s.c.)(7) and SnPP (20 μmol/kg B.W., s.c.)(52). The magnitude of hepatic HO inhibition reported here agrees well with that described in suckling rats administered 40μmol/kg B.W. ZnPP twice daily for 2 d(26), and in neonatal rats after a 1 μmol/kg B.W., s.c. postnatal dose of SnMP(7). Splenic HO activity remained largely unaffected by ZnPP administration, consistent with observations in neonatal rats after antenatal SnPP exposure(18). This may be due to low splenic ZnPP concentrations, rather than a refractoriness to inhibition, because splenic HO activity was reduced in suckling rats administered 40μmol/kg B.W. ZnPP twice daily for 2 d(26).

The HO inhibition observed between d 1 and 4 after dosing was also consistent with the decreased serum bilirubin levels observed during this same time period. The lack of significance after d 1 is most probably due to the short duration of the mild hyperbilirubinemia observed in neonatal rats(6). Furthermore, serum bilirubin levels are the net result of plasma clearance, conjugation, excretion, and production, and therefore, may not accurately reflect changes in bilirubin production alone(1). The pattern of bilirubin reduction was similar to that observed for SnMP (1 μmol/kg, at birth)(7), but shorter than that reported for SnPP (five sequential doses of 100 μmol/kg B.W. beginning at birth) in neonatal rats(6), probably due to differences in dosing regimens.

Maximal ZnPP concentrations in liver, spleen, and kidney were achieved within 2-4 d after dosing. This pattern was similar to that observed in adult rats after ZnMP administration (15 μmol/kg B.W., i.v.)(27), but differed from that reported for SnPP and SnMP. Specifically, although the liver was the major site of ZnPP deposition, the kidney and liver were the predominant SnPP and SnMP accumulation sites in adult rats dosed at 10-50 μmol/kg B.W.(7, 52). However, this may be dose-dependent, as the liver was the major deposition site for SnMP at lower doses in adult rats, and for SnPP in neonatal rats after antenatal exposure to doses below 50 μmol/kg B.W.(7, 18). In addition, the hepatic ZnPP concentrations reported here are 5-10-fold greater than SnPP concentrations observed in neonatal rats exposed antenatally(18). As reported after SnPP dosing in adult rats(52), ZnPP was retained by the neonatal rat spleen during subsequent organ development. The reason for this is unknown, but may indicate a reduced capability to clear these compounds. It seems unlikely that this is related to erythrocyte ZnPP levels, because the latter constituted only 10% of the nanomoles of ZnPP found in the spleen.

In these experiments, neonatal rat brain ZnPP concentrations were not significant and were severalfold lower than those reported for SnPP in neonatal rats administered 50 μmol/kg B.W. s.c. at birth(18). The fact that ZnPP could barely be detected in the brain under these dosing conditions suggests that ZnPP does not readily cross the blood-brain barrier in significant quantities. This is consistent with the lack of brain HO inhibition after ZnPP dosing reported in earlier studies(19, 31) and the low brain concentrations found in adult rats after ZnMP dosing(27). Therefore,in vivo ZnPP treatment should not impact on neuronal CO production or guanylate cyclase activity in the brain, as reported in in vitro studies(53, 54). This may be important clinically as one potential toxicity of metalloporphyrin administration is the inhibitory effect on these heme-related neural second messenger pathways.

In agreement with a previous study by Qato and Maines(31), the ZnPP content of circulating neonatal rat erythrocytes, as determined by analysis of erythrocyte zinc by atomic absorption spectroscopy, increased above basal levels. Whether this represents direct incorporation of administered ZnPP or increased de novo ZnPP synthesis in vivo is unknown. However, in contrast to the results obtained in the previous study with newborn nonhuman primates, neonatal rat erythrocytes did not constitute a major deposition site under these experimental conditions.

The total ZnPP recovered at any one time point in the blood and organs studied accounted for less than 10% of the administered dose. This low recovery is consistent with results obtained with other metalloporphyrins administered to adult rats. For example, Anderson et al.(52) reported recoveries of 15-30% from 10 tissues between 12 and 48 h after SnPP administration (10 μmol/kg B.W., i.v.). In addition, the low hepatic and splenic concentrations found at 24 and 48 h after ZnMP dosing (15 μmol/kg B.W., i.v.) suggests an even lower percent recovery(27). It seems unlikely that the low recovery is due to HO metabolism of ZnPP or a pronounced photolability of the compound, because we can account for approximately 80% of an i.v. administered ZnPP dose in adult rats (our unpublished observations). It may be that ZnPP was slowly removed from the i.p. injection site to the liver and rapidly excreted in the bile. This is consistent with the reported biliary excretion of ZnPP by Qato and Maines(31). In addition, preliminary studies conducted in neonatal rats indicated that intestinal tissue samples contained significant amounts of ZnPP (data not shown).

Levels of hepatic HO-1 mRNA and HO-1 protein transiently increased in neonatal rats after i.p. ZnPP dosing. Although metalloporphyrin-induced changes in HO-1 have not been observed previously in neonatal animals, this result is consistent with the increased hepatic HO-1 protein and mRNA levels observed in adult rats after SnPP or ZnPP dosing(20, 23, 55). Differences in route of administration probably explain why this result differs from the observations of Lin et al.(55) in neonatal rats after antenatal SnPP exposure. Regardless, the slight increase in HO-1 protein levels observed only on d 1 was not sustained. Therefore, it is unlikely that it was a confounding factor in the duration of HO inhibition or in the reduction of serum bilirubin levels.

In summary, our results demonstrate ZnPP has a fairly short duration of action in neonatal rats of between 4 and 7 d, when administered i.p. as a single dose at 40 μmol/kg B.W. In addition, ZnPP treatment also failed to elicit certain potentially adverse affects observed with other metalloporphyrins, such as weight loss and growth retardation(56), and significant deposition in the brain. Currently, only limited data are available regarding the use of metalloporphyrins for the prevention of neonatal jaundice. Concern regarding the safety of the administration of metalloporphyrins remains despite the early successful results(9, 10), and a debate still exists regarding which metalloporphyrin should be used. In this context, our results derived in newborn rats offer some support for considering ZnPP as an alternative drug, once its safety has been clearly established.