Toxicological Sciences 62, 80-91 (2001)
Copyright © 2001 by the Society of Toxicology
REPRODUCTIVE AND DEVELOPMENTAL TOXICOLOGY |
Growth and Development in Rats Given Recombinant Human Epidermal Growth Factor1-48 as Neonates
Department of Pathology and Experimental Toxicology, Parke-Davis Pharmaceutical Research Division, Warner-Lambert Company, Ann Arbor, Michigan 48105
Received June 23, 2000; accepted February 27, 2001
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To assess effects of supraphysiologic doses of human recombinant epidermal growth factor1-48 (rhEGF1-48) on neonatal rats, 10 litters of Wistar rats/treatment group were given 0 (formulated vehicle), 10, 100, or 1000 µg/kg daily by subcutaneous injection on postnatal days (PND) 1 through 6. Clinical signs, body weight, acquisition of developmental landmarks and reflexes, and behavior were monitored during treatment and for 5 weeks thereafter (to PND 42). A subset of animals was euthanized weekly from PND 7–28 and necropsied. Selected tissues were examined microscopically. Body weight gain at 1000 µg/kg during treatment was significantly less than control. Precocious incisor eruption, eye opening, vaginal opening, and preputial separation occurred at 100 and/or 1000 µg/kg. Acquisition of reflexes (negative geotaxis, wire maneuver, acoustic startle reflex, and visual placing) was delayed at 1000 µg/kg. Acquisition of adult locomotion was also delayed at 1000 µg/kg. These effects were transient, as locomotor activity at PND 28 and 42 did not differ from control. Effects on acoustic-startle responding persisted in females to final assessment on PND 42. Habituation to repeated acoustic stimuli was impaired, as well as response inhibition following a prepulse acoustic stimulus. rhEGF1-48 induced structural changes in the skin, retina, kidney, oral and nasal mucosa, lung, and liver. Many of these changes were consistent with the expected mitogenic activity of rhEGF1-48 and were transient in nature, as severity and incidence diminished with time. An exception was changes observed in the retina at 1000 µg/kg (rosettes/folds and focal defects in the outer nuclear/photoreceptor layers) that were still present 3 weeks after termination of treatment. Acceleration of developmental landmarks; suppression of reflexes, behavior, and somatic growth; and mitogenic responses in epidermal tissues have been reported in rodents treated with epidermal growth factor (EGF) derived from various mammalian species. These results demonstrate that a 48-amino acid fragment of human EGF produced by recombinant technology also induces such effects.
Key Words: rhEGF1-48 toxicity; human epidermal growth factor1-48; neonates; rats; development; behavior..
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recombinant human epidermal growth factor1-48 (rhEGF1-48) is a 48-amino acid fragment of human epidermal growth factor (EGF), produced in Escherichia coli using recombinant DNA technology. rhEGF1-48 is a potent mitogen in vitro and retains the full spectrum of biological activity of the 53-amino acid endogenous form of human EGF in the absence of the final 5 carboxy-terminus amino acids (unpublished data, Parke-Davis Pharmaceutical Research). A variety of pharmacological actions have been attributed to EGF, including stimulation of cell growth in vitro and in vivo, inhibition of gastric-acid secretion, stimulation of ulcer healing, and stimulation of prostaglandin-E2 synthesis (Carpenter and Wahl, 1990
). EGF has been shown to promote gastrointestinal mucosa regeneration/healing (Guglietta and Sullivan, 1995; Konturek et al., 1988; Musroe et al., 1991), leading to the proposal that EGF might be useful in the treatment of gastrointestinal erosive/ulcerative disorders, including necrotizing enterocolitis in infants. Recombinant human EGF (100 ng/kg/h) was given to an 8-month-old child with necrotizing enteritis, and resulted in histological repair of intestinal mucosa and clinical recovery in approximately 2 months (Sullivan et al., 1991).
The peptide fragment rhEGF1-48 induces intestinal cell proliferation (Haskins et al., 1995, Haskins et al., 1997) and may have potential use in infants. In addition to the aforementioned pharmacological actions in adults, EGF also appears to play an important role in regulation of cell proliferation and differentiation during development. Various forms of EGF administered to neonatal rodents produce both accelerating and retarding effects on somatic and behavioral development (Calamandrei et al., 1993). Subcutaneous injection of mouse EGF in neonatal rats and mice produces alterations in craniofacial development characterized by precocious eye opening and incisor eruption. However, retardation of somatic growth, inhibition of hair growth, and delayed development of some reflexes and behaviors have also been observed in the same animals (Calamandrei and Alleva, 1989; Cohen, 1962; Hoath, 1986; Smart et al., 1989; Tam, 1985). Because of the potential for clinical use of rhEGF1-48 in infants, it was considered necessary to determine in neonates if pharmacological responses during development were similar to those of other forms of EGF, and to explore the potential for unexpected toxicity. The present study was therefore undertaken to evaluate potential effects on growth and development induced by administration of rhEGF1-48 to neonatal rats.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animals.
The rat was selected for this study because the biological activity of various forms of EGF, including human EGF, has been well characterized in this species. Forty timed-pregnant female Wistar rats [Crl:(WI)BR VAF/Plus®; Charles River Breeding Laboratories, Inc., Kingston, New York], obtained on gestation day (GD) 13, were allowed to deliver their offspring. The day of delivery was considered postnatal day (PND) 0. Each dam was housed individually (except during lactation) in a stainless steel, hanging wire-mesh cage. Near the time of parturition, a solid stainless steel plate and bedding were added to the home cage. Following weaning on PND 21, surviving male and female offspring were segregated by sex into separate cages according to litter. Each dam and her offspring were uniquely identified. Food (Purina Certified Lab Rodent Chow® No. 5002, Ralston Purina Co., St. Louis, MO) and water were available ad libitum throughout the study. Environmental conditions were in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996).
On GD 20, dams with their respective litters were randomly assigned to 4 experimental groups (10/group) and, although dams remained untreated, each litter was treated with vehicle or rhEGF1-48 at 10, 100, or 1000 µg/kg. All offspring were evaluated for acquisition of developmental landmarks. Offspring were assigned to subgroups (in general composed of 7–10 rats/sex/group) for reflex and behavior testing, or for pathologic evaluation. The first male and female of each litter were assessed for acquisition of reflexes and behavioral parameters. No more than one rat/sex/litter was evaluated for these parameters. The subsequent 5 males and females of each litter were designated for euthanasia at weekly intervals for evaluation of pathologic changes.
Test material.
Recombinant human epidermal growth factor1-48 (rhEGF1-48), purified from Escherichia coli, is a fragment of the 53-amino acid human EGF (hEGF1-53). The amino acid sequence is Asp(NH2)-Ser-Asp-Ser-Glu-Cys-Pro-Leu-Ser-His-Asp-Gly-Tyr-Cys-Leu-His-Asp-Gly-Val-Cys-Met-Tyr-Ile-Glu-Ala-Leu-Asp-Lys-Tyr-Ala-Cys-Asp(NH2)-Cys-Val-Val-Gly-Tyr-Ile-Gly-Glu-Arg-Cys-Glu(NH2)-Tyr-Arg-Asp-Leu-Lys-OH. It lacks the final 5 carboxy terminus amino acids of hEGF1-53, Trp-Trp-Glu-Leu-Arg(COOH). rhEGF1-48 was reconstituted in a vehicle of 20 mM phosphate buffer containing 0.01% Tween 80 (pH 6.0) and was dosed based on active moiety (49.1 µg/ml).
Reconstituted rhEGF1-48 was further diluted with sterile 5% dextrose in water (D5W), with a final concentration of 0.5-mg rat albumin/ml, to obtain solutions of 0.2, 2, and 20 µg/ml at 10, 100, and 1000 µg/kg, respectively. Control animals received formulated vehicle diluted with D5W and rat albumin in the same ratio as animals administered 1000 µg/kg. Dosing solutions were filtered through a 0.2-µm filter and were transferred to a sterile container.
Samples from each dosing solution were analyzed for drug concentration by an HPLC assay and were found to be within 10% of the intended value, with the exception of the 10 µg/kg dosing solution, which was below the limit of detection. Mitogenic activity of each dosing solution was assessed pre- and post-dosing, and was within the acceptable range of biologic activity for the assay.
Treatment.
Offspring received solutions of rhEGF1-48 or vehicle by subcutaneous injection in the intrascapular region, once daily, at a dose factor of 50 ml/kg of body weight from PND 1 (approximately 24 h after completion of delivery) through 6. A neonatal treatment period was found to induce maximal biological activity in previous studies with EGF in rats. Dose volumes were based on the most recent individual body weights.
Doses were selected based on preliminary results of an exploratory study with rhEGF1-48 in adult male Wistar rats, as well as on information from the scientific literature on various forms of EGF administered to rodents. In the exploratory study, 100 or 1000 µg/kg administered as a single intravenous injection or as a continuous infusion via a subcutaneously implanted mini-pump were well tolerated. Subcutaneous injection of mouse EGF has been shown to produce precocious eye opening and incisor eruption, but also retardation of somatic growth, as well as some reflexes and behaviors, in neonatal mice at 2700 to 4000 µg/kg/day (Calamandrei and Alleva, 1989; Tam, 1985), and in neonatal rats at 500 µg/kg/day (Hoath, 1986). A comparative study of mouse EGF to human EGF also revealed accelerated eye opening in neonatal mice at subcutaneous doses of 250 to 2000 µg/kg/day for both compounds (Smith et al., 1985). Based on these results, doses of 10, 100, and 1000 µg/kg/day were selected.
Each dam was handled daily following arrival in the laboratory in an attempt to minimize maternal stress and any ensuing effects on offspring during the postnatal period. Offspring were observed daily throughout the study for clinical signs and survival, and body weights were recorded. All offspring were retained (i.e., litter sizes were not standardized) to provide a sufficient number to fill all subgroups.
Developmental landmarks, reflexes, and behavior.
The developmental landmarks, reflexes, and behavioral parameters indicated in Table 1 were evaluated from the PND listed until acquisition. For developmental landmarks, acquisition occurred when both pinnae were detached and both eyes were open, when both lower incisors erupted, and when the vaginal opening became patent. Preputial separation was determined according to the method of Korenbrot et al. (1977). Evaluation of the static righting reflex, wire maneuver, visual placing, and negative geotaxis was according to standard methodology (Irwin, 1968; U.S. EPA, 1985; Moser et al., 1988). Auditory startle was evaluated by placing the animal in a sound-attenuating enclosure; an auditory stimulus was elicited by a hand clicker, and a positive response was recorded if flicking of the pinnae and/or a sudden jerk of the body was observed.
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Offspring were assessed for acquisition of 4 unique stages of locomotion, according to the method of Vorhees and coworkers (Vorhees et al., 1979
). Locomotion was monitored from the most primitive form, pivoting (Stage 1), to forward locomotion with the head low and body low (Stage 2), to forward locomotion with the body raised and head low (Stage 3), to adult locomotion with the head and body raised (Stage 4). Acquisition of each of these 4 stages was analyzed separately.
On PND 28 and 42, each animal was monitored for locomotor activity and habituation to a novel environment during a 10-min test session in an automated activity monitor (Digiscan Activity Monitor System with OASIS software, AccuScan Electronics, Inc., Columbus, OH). Prior to testing, diagnostic and calibration procedures were conducted on the activity monitor system, as described previously (Henck et al., 1996). The duration of the test session was considered sufficient to evaluate the initial response to the novel environment of the activity monitor during test minute 1, as well as to observe the rate in decline of activity (considered a measure of habituation) over 10 consecutive 1-min test periods. Five parameters (total distance, number of vertical movements, rest time, stereotypy time, and center time) were considered to be indicative of the spontaneous movement of a rat and were subjected to group-mean comparison.
On PND 42, following activity monitor testing, each animal was evaluated for the acoustic startle response, as well as prepulse inhibition and habituation to an acoustic stimulus. Prior to testing, calibration and sensitivity evaluation procedures were conducted on the acoustic startle system, as described previously (Henck et al., 1996). Each animal was given 70 trials, using the SR-LAB Startle Response System (San Diego Instruments, San Diego, CA). The trials alternated between a background noise of 70 dB (total of 30 trials), a noise-level (NL) tone of 120 dB (total of 20 trials), and a prepulse (PRE) tone of 90 dB, followed in 100 ms by a noise level tone of 120 dB (total of 20 trials), separated by intertrial intervals ranging from 5 to 30 s. Peak response (maximum input voltage) for the first NL and PRE trials, the rate of habituation over 20 NL trials, and percent response inhibition resulting from the prepulse tones were evaluated.
Pathology.
On PND 7, 14, 21, and 28, 1 animal/sex/litter (if available) from each treatment group was anesthetized with ether and decapitated. Major tissues and organs were examined grossly. Formalin-fixed brain, kidneys, small and large intestines, and liver were weighed from animals in the first 5 litters/group. To assess intestinal length, small and large intestine from these animals were extended to full length, and measured. Brain, liver, kidney, stomach, small and large intestine, and skin were fixed in 10% buffered formalin. Eyes were fixed in 6% glutaraldehyde. Formalin-fixed tongue, nasal cavity, hard palate, trachea, mandibular salivary gland, esophagus, urinary bladder, lung, and thyroid were collected on PND 7 only, from 1 animal/sex/litter from the first 5 litters/group in the control and 1000 µg/kg group. All tissues were processed in paraffin, stained with hematoxylin and eosin, and examined microscopically.
Statistical analysis.
To control for the multiplicity of statistical comparisons (i.e., reduce the number of false positive conclusions), all sets of developmental and behavioral response measures (parameters) were divided into distinct classes based on the relationship of the parameters. For example, all developmental landmark data comprised one class, while all activity monitor data on a specific day comprised another. The level of significance for each comparison within the class was 0.05 divided by the square root of the number of parameters in the class (Tukey et al., 1985), to provide an approximate classwise significance level of 5%. Organ weight data were not partitioned and were analyzed at an approximate class-wise significance level of 1%.
The basic trend test employed was the sequential trend test, using the rank-dose scale and rank-transformed data (Park, 1985; Tukey et al., 1985). This test is equivalent to sequential application of the Kruskal-Wallis 1-way analysis of variance (ANOVA) by ranks (Kruskal and Wallis, 1952), with the treatment effects being evaluated by dose-trend tests that have contrast coefficients for equally spaced (ranked) treatment groups. The sequential linear trend test is designed to detect monotone changes in dose response; it does not detect trend reversal or curvature. If the true dose response was not monotonic, the trend test was considered not sensitive enough to detect the treatment effect. A trend reversal test was then conducted, consisting of a quadratic trend test performed at the 2-tailed 1% class-wise significance level. If the trend reversal test was significant and the high-dose trend test was not significant, the treatment groups were compared to control by Dunnett's test (Dunnett, 1955, Dunnett, 1964), using rank-transformed data, and performed at the 5% class-wise significance level.
The monotonic dose-response relationship tested by the trend test was not considered realistic for the activity monitor parameters, because they have been demonstrated with several drugs to exhibit a U-shaped dose-response curve (Iversen and Iversen, 1981). Hence, Dunnett's test on rank-transformed data was performed in place of the trend test as the main analytical method. Acoustic startle data, as well as the activity monitor parameters total distance and number of vertical movements, were subjected to profile analysis (Johnson and Wichern, 1982) to evaluate the response by treatment group interaction (parallelism test) and, secondarily, to address the question of equal group effects; the raw data were used for this analysis. The methods of Greenhouse and Geisser (1959) were incorporated in the profile analysis to produce conservative tests for the parallelism hypotheses.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Clinical Observations and Growth
Generalized exfoliation occurred over the entire body in approximately 70% of animals at 1000 µg/kg during the week following treatment. Treatment-related ocular alterations occurred in approximately 20% of animals at 1000 µg/kg during this period, and included corneal drying or opacity, enlarged eyelid, keratitis sicca, and unilateral eye enlargement; these observations appeared a few days after precocious eye opening.
Eight of 103 animals at 1000 µg/kg died or were euthanized moribund. No deaths or treatment-related clinical signs occurred at 10 or 100 µg/kg.
Body-weight gain during the treatment period at 1000 µg/kg was less than control by 23% and 27% for males and females, respectively. No treatment-related body weight changes were apparent at 10 or 100 µg/kg throughout the study or at 1000 µg/kg during the post-treatment period (PND 7–42).
Developmental Landmarks and Reflexes
Significantly (p < 0.025) accelerated acquisition of lower incisor eruption (by 1–2 days), eye opening (by 3–5 days), and preputial separation (by 4–7 days) occurred at 100 and 1000 µg/kg, and of vaginal opening (by 12 days) occurred at 1000 µg/kg (Table 2). Timing of pinnae detachment was not affected by treatment.
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Development of all reflexes evaluated, with the exception of static righting reflex, was significantly (p < 0.025) delayed in males and/or females at 1000 µg/kg (Table 3
): negative geotaxis (by 2 days), acoustic startle (by 1–2 days), wire maneuver (by 2 days), and visual placement (by 1.5 days).
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Behavior
No treatment-related effects were seen on initial stages of locomotor development. Progression to the final stage of adult locomotion was delayed by 1.5–2 days in males and females at 1000 µg/kg (Fig. 1
), although this change was not statistically significant. Evaluation of locomotor activity in an automated activity monitor on PND 28 and 42 revealed no treatment-related effect on horizontal or vertical activity, stereotypy, or habituation to the novel environment of the open field (data not shown).
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The acoustic startle response to 120 dB tones (evaluated as maximum input voltage) for males at 10 µg/kg, but not at 100 or 1000 µg/kg, was less than control during the majority of 20 trials, by a maximum of 52% (Fig. 2
). However, because maximum input voltage for many of these trials was within the historical control range for this laboratory, and because no dose-response relationship was evident, this apparent effect was considered not biologically significant. Group mean maximum input voltage for females at 1000 µg/kg was comparable to control for the first 10–120 dB trials; however, for the next 10 trials, maximum input voltage was up to 2-fold greater than control. Although comparison across the 20 trials revealed no statistical significance, the mean and standard error for maximum input voltage of females at 1000 µg/kg during the majority of the last 10 trials was greater than the mean and standard error of control females, and observation of the data indicated that responding did not diminish with repeated acoustic stimuli (Fig. 2). Group mean percent response inhibition following a prepulse stimulus for 20–120-dB and 20 prepulse trials is given in Table 4. Although no statistically significant differences were apparent for maximum input voltage for the first prepulse trial for males or females, group mean and standard error percent response inhibition of females at 1000 µg/kg was less than the mean and standard error of controls.
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Pathology
Organ weight data and histopathologic findings are summarized in Tables 5 and 6
, respectively. Alterations were noted in skin, eye, kidney, nasal and oral mucosa, lung, and liver of male and female rhEGF1-48-treated rats terminated at various intervals.
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The skin of rhEGF1-48-treated animals had dose-related increases in the incidence and intensity of epidermal and follicular changes. Epidermal hyperkeratosis and acanthosis, and a decreased proportion of anagen hair follicles relative to catagen/telogen follicles occurred at all doses on PND 7 (Fig. 3
). These changes were generalized, involving intrascapular (injection site) and flank skin at 1000 µg/kg. Epidermal hyperkeratosis accounted for skin scaling (exfoliation) noted clinically. On PND 14, the proportion of anagen to catagen/telogen hair follicles was reduced at 100 and 1000 µg/kg, although the incidence of this finding was not as great as on PND 7. This finding corresponded to the clinical observation of thin hair coats at 1000 µg/kg. By Day 14, hyperkeratosis was still evident at 1000 µg/kg, and acanthosis was present in only one animal. Two 1000-µg/kg animals had increased numbers of distorted or deformed hair follicles. On PND 21, an increase in the proportion of catagen and anagen hair follicles relative to telogen follicles was apparent in rhEGF1-48-treated rats at all doses. Telogen follicles predominated in controls at this time. By PND 28, the skin of all but one rat at 1000 µg/kg was indistinguishable from control.
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Retinal and corneal lesions were first evident on PND 7 and 14, respectively. On PND 7, the retinal nuclear layers of rats at 1000 µg/kg were diffusely hypocellular and/or had increased mitoses in the outer nuclear layers (delayed maturation). The incidence of developmental malformations of the retina, classified as rosettes/folds (peripheral and central), was also increased at 1000 µg/kg on PND 7 through 28. Additional retinal malformations, consisting of cell nuclei from the outer nuclear layer in the photoreceptor layer, occurred in several animals at 1000 µg/kg on PND 14 and 21 (Fig. 4
). Corneal neovascularization, neutrophilic inflammation, and reactive epithelial hyperplasia were noted on PND 14 at 1000 µg/kg. Corneal findings corresponded to dry corneas noted grossly in several rats. Neovascularization and neutrophilic inflammation were still present in some animals at 1000 µg/kg terminated on PND 21 and/or 28.
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Hypercellular epithelial cell foci, reminiscent of immature renal tubules, were observed in the renal cortex of rats at 1000 µg/kg on PND 14–28. Multifocal basophilic renal tubules, indicative of slight tubular degeneration occurred with increased incidence and frequency at 1000 µg/kg in rats terminated on PND 14–28.
Diffuse epithelial hyperplasia, hypertrophy, and hyperkeratosis were apparent in the lingual and palatine mucosa of all animals at 1000 µg/kg (Fig. 5). Mucosal thickness was at least 1.5- to 2-fold greater than control. Nasal respiratory epithelium was also hyperplastic at 1000 µg/kg (Fig. 6). Respiratory mucosal height was increased and epithelial cells were hypertrophic; goblet cells were increased in size and number within the pseudostratified epithelium. In lung, the cellularity of alveolar septa of rhEGF1-48-treated animals was reduced compared to controls at 1000 µg/kg (Fig. 7). Furthermore, alveoli were larger in size and septal development appeared to be at a more advanced stage than in controls.
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A slight increase in intensity and incidence of microvesicular cytoplasmic vacuolation of hepatocytes occurred at 1000 µg/kg on PND 7 and correlated with pale livers noted grossly. Liver weight relative to body weight was significantly increased in females at 1000 µg/kg. Histological correlates for the significant increase in small intestinal weight in males and females at 1000 µg/kg were not apparent, and no treatment-related changes were apparent in the length of the small orlarge intestines. Drug-related microscopic alterations were not evident in stomach or small and large intestines.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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rhEGF1-48-associated clinical observations occurred only at 1000 µg/kg and were either expected pharmacological effects related to the skin (du Cros, 1993
) or considered a consequence of precocious eye opening. Body weight-gain suppression was consistent with previous reports in which rodents treated as neonates with various forms of EGF had growth retardation (Calamandrei and Alleva, 1989; Cohen, 1962; Hoath, 1986; Tam, 1985). Precocious incisor eruption and eye opening were also expected, based on previous reports (Calamandrei and Alleva, 1989; Cohen, 1962; da Silva et al., 1991; Hoath, 1986; Smart et al., 1989; Tam, 1985). Pinna detachment was not delayed in the present study, as was observed in previous studies, likely due to timing differences. In the present study, treatment was initiated on PND 1, rather than on PND 0, as with previous studies.
Cleavage of the balanopreputial gland and vaginal opening, considered indicators of puberty in the rat, were accelerated in rhEGF1-48-treated animals. The influence of EGF on cleavage of the balanopreputial gland has not been reported previously. Precocious vaginal opening has been reported in hamsters given subcutaneous injections of human EGF during the second postnatal week (advanced by 3 days) and in rats given subcutaneous injections of human EGF during late gestation (advanced by 5–6 days) (da Silva et al., 1991; Smart et al., 1989). Absence of effects on ovarian and uterine weights on the day of vaginal opening in EGF-treated rats suggested that advancement of this developmental landmark was not due to sexual maturation, but rather to the altered development of the perineal epithelium (da Silva et al., 1991). This idea was supported by the work of Nelson et al. (1991), who determined that 750 ng mouse EGF implanted in a slow-release capsule in ovariectomized adult mice induced vaginal keratinization. Similar effects induced by estrogen were inhibited by an EGF-specific antibody, implicating EGF as a mediator of estrogenic action in mice. Vaginal opening occurred earlier following neonatal treatment in the present study than in previous rodent studies, which treated subjects during late gestation or the second postnatal week. These differences suggest that the degree of acceleration of vaginal opening depends on the developmental stage of exposure.
Delayed acquisition of reflexes, as noted in the present study, has been reported in studies with several forms of EGF (Calamandrei et al., 1989; Smart et al., 1989; da Silva et al., 1991). Delayed acquisition of the acoustic startle reflex is of particular interest because altered responses were still detected in females at PND 42. These effects in young adults were manifested as failures to reduce responding to repeated acoustic stimuli, and to inhibit responding following a prepulse tone. Failure to decrease responding to repeated stimuli might be the result of disruption of habituation (considered a measure of simple learning), or might result from enhanced reactivity to stimuli in general. However, if animals were hyperreactive, their response to any type of sensory stimuli might be greater than expected, and this was not observed clinically, or in initial acoustic startle trials. Response inhibition following a pre-pulse tone was less in females at 1000 µg/kg. Prepulse inhibition is considered a more sensitive indicator of ototoxic damage than is the response elicited by a single auditory stimulus (Young and Fechter, 1983), and is used as a means of distinguishing hearing deficits from motivational and neuromuscular deficits (Fechter and Young, 1983). It is unlikely that significant motivational and neuromuscular deficits contributed to these acoustic startle effects. Although acquisition of the various stages of locomotion appeared to be delayed by treatment with rhEGF1-48, testing of spontaneous locomotor activity at 28 and 42 days of age revealed no treatment-related effects, underscoring the transient nature of the delay. Although these results indicate that some aspect of acoustic startle responding is disrupted in female rats treated with rhEGF1-48 as neonates, the exact meaning of these effects, or their relationship to developmental delays, cannot be ascertained from this screening test. Further exploration of this effect would require the use of different sensory modalities and a more stringent evaluation of prepulse inhibition.
Skin changes noted in rhEGF1-48-treated animals were consistent with those reported previously for neonatal rats and mice given epidermal growth factor (du Cros, 1993). The sequential changes in hair-follicle development were suggestive of delayed follicular development. That the skin of most rhEGF1-48-treated animals on PND 28 was morphologically indistinguishable from controls indicates that the drug-induced skin changes were largely transient and fully reversible following cessation of treatment.
Focal defects in the outer nuclear/photoreceptor layers of the retina and the increased incidence of retinal rosettes/folds at 1000 µg/kg indicate that rhEGF1-48 may have direct or indirect effects on retinal development. These changes were still apparent in some animals on PND 28 and were therefore not totally reversed. Increased mitoses in the outer nuclear layer and hypocellularity of the retina in several animals at 1000 µg/kg on PND 7 may indicate a delay in retinal development. These latter changes were not apparent at later time points, suggesting a transient response. The cause of the corneal alterations at 1000 µg/kg was not clear. Premature opening of the palpebral fissure may have predisposed the cornea to irritation or drying.
Basophilic renal cortical tubules associated with hypertrophic epithelial cells and sporadic evidence of cell degeneration occurred in treated and control animals. Tubular dilatation also occurred sporadically in basophilic tubules. Basophilic tubules were slightly more common in animals treated with rhEGF1-48. These appeared to be persistent foci of developing tubules and may have been a consequence of a transient delay in renal maturation. Significant growth retardation of the kidney has also been observed in weanling rats treated with mouse EGF on PND 0–3 (Tam, 1985).
Hyperplastic changes in tissues of the oral cavity have previously been documented in EGF-treated mice (Steidler and Reade, 1981). Changes in the nasal respiratory epithelium have not been reported previously in EGF-treated neonates, but are consistent with those identified in adult rats given rhEGF1-48 for 4 weeks by continuous infusion (Breider et al., 1996). Pulmonary changes in rhEGF1-48-treated neonates in this study were suggestive of enhanced or accelerated maturation of peripheral lung parenchyma, and are consistent with findings previously observed in several animal species treated with EGF during fetal stages of development (Catterton et al., 1988; St. George et al., 1991; Sundell et al., 1980).
The significance of the pale livers attributed to increased microvesicular cytoplasmic vacuolation of hepatocytes in treated animals is uncertain. This change was only noted in rats euthanized on PND 7, and was not associated with other degenerative or proliferative changes. Cytoplasmic vacuolation also occurred to a lesser degree in several control animals. This change, reflecting fat accumulation in hepatocytes, may have been a consequence of the greater interval these animals were away from the dams on the day of euthanasia and consequent inanition resulting in immobilization of peripheral fat stores to the liver (Hathcock, 1985), rather than a drug-related effect. Fatty vacuolation of hepatocytes has been previously reported in EGF-treated neonatal mice (Heinberg et al., 1965).
It is difficult to distinguish whether many of the pathologic observations were directly attributable to EGF effects on specific tissues, or were due to indirect effects secondary to growth retardation or EGF-related changes in cardiovascular parameters.
Results of this study indicate that treatment of neonatal rats with the EGF fragment, rhEGF1-48 resulted in growth retardation, accelerated developmental landmarks, and delayed acquisition of reflexes at 1000 µg/kg, and mitogenic responses in epidermal tissues at 
10 µg/kg. These changes correspond to those resulting from neonatal administration of other forms of EGF to rodents. With the exception of effects on acoustic startle responding and changes in the retina, treatment-related findings were considered transient.
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ACKNOWLEDGMENTS |
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The authors thank Denise Frahm and Michelle Cole for technical expertise, Walt Bobrowski for preparation of photomicrographs, Jamie Colgin and Joyce Zandee for statistical analyses, and Dr. Robert Parker for peer review of histopathologic data.
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NOTES |
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1 To whom correspondence should be addressed at: Eli Lilly and Company, P.O. Box 708, Drop Code GL43, Greenfield, IN 46140. Fax: (317) 277-7601. E-mail: henck_judith_w{at}lilly.com.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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