| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online January 5, 2005 as doi:10.1096/fj.04-2550fje. |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Laboratories of Biochemistry, University of Pennsylvania School of Veterinary Medicine, Philadelphia, Pennsylvania, USA
1Correspondence: Laboratories of Biochemistry, School of Veterinary Medicine, University of Pennsylvania, 3800 Spruce St., Philadelphia, PA 19104-6048, USA. E-mail: shapirob{at}vet.upenn.edu
SPECIFIC AIMS
Perinatal exposure to phenobarbital produces a multitude of permanent reproductive, growth, locomotor, and learning dysfunctions in animals and humans. In addition, affected individuals exhibit postpubertal above-normal multicytochrome P450-dependent hepatic drug metabolism. We administered phenobarbital to newborn rats at a therapeutic-like anticonvulsant dose, considerably below the threshold for inducing morphologic and behavioral defects, to determine whether early exposure to the barbiturate permanently alters (or imprints) constituent expression as well as the inductive response of hepatic isoforms of cytochrome P450 (P450, CYP) to environmental-like levels of inducing agents leading to an imperceptible but pernicious accumulation of toxic metabolites, enhancing tumorigenesis and reducing life expectancy.
PRINCIPAL FINDINGS
1. Neonatal phenobarbital increases tumor formation, urinary tract pathologies, and reduces life span
Neonatal exposure to phenobarbital resulted in a small (
4%), occasionally significant (P <0.05) reduction in body weights of males and females throughout the 2 year study. However, concurrent determinations of linear growth and obesity (Lee index) indicated no effect of the treatment. Organ weights (liver, kidneys, adrenals, seminal vesicles, or uterine) at 2 years of age were statistically indistinguishable from vehicle control rats of the same sex. In contrast, a statistically larger number (P <0.01) of barbiturate-exposed rats of both sexes died at a younger age than the controls, resulting in a reduced mean life expectancy (Table 1
). At the time of death, necropsy results demonstrated a >2-fold increase in the incidence of tumors in barbiturate-exposed rats compared with same-sex controls (Table 1)
. The high incidence of mammary tumors in senescent female rats was responsible for a dramatic sexual dimorphism in tumor numbers. In contrast, no blood was found in the urine of females at the time of death but was apparent in 10% of vehicle-treated and 37% of phenobarbital-treated males (P <0.05), indicating an enhanced susceptibility to kidney or urinary tract pathologies in old male rats neonatally exposed to phenobarbital.
|
2. Senescent (2 years old) rats neonatally exposed to phenobarbital express above-normal levels of total P450, cytochrome b5, and hexobarbital hydroxylase as well as constitutive P450 isoforms CYP2C6 and CYP2C7
The broad determinants of in vitro drug metabolism (cytochrome b5, total P450, and hexobarbital hydroxylase) were found at significantly greater concentrations (10%, 15%, and 45%, respectively) in the livers of aged male and female rats neonatally exposed to phenobarbital than in comparable age- and sex-matched rats neonatally exposed to the vehicle. However, the so-called drug metabolizing enzymes are functional and not structural entities whose activities are dependent on the contribution of various isoforms of P450, each with a different affinity for the drug substrate. The near dozen hepatic isoforms of P450, each contributing at various degrees to the metabolism of different drugs as well as endogenous substrates (e.g., steroid hormones, prostaglandins, fatty acids, etc.) are divided into 2 classes: constitutive (always expressed) and inducible (not expressed in untreated rats). Measuring expression levels of major constitutive isoforms, we observed a permanent 30–40% overexpression (P <0.01) of CYP2C7 in 2-year-old males and females neonatally exposed to phenobarbital. In contrast, neonatal exposure to the barbiturate produced persistent above-normal (30%) expression levels of CYP2C6 in treated females only.
To demonstrate that the phenobarbital imprinted overexpression of multi-P450-dependent drug metabolizing enzymes and specific P450 isoforms was not simply a curious "test tube" phenomenon, we measured the pharmacologically functional endpoint of hexobarbital-induced sleep times. If the permanent overexpression of hexobarbital hydroxylase and contributing P450 isoforms was biologically significant, we expected to observe a commensurate decline in hexobarbital-induced sleep times. We did observe a permanent postpubertal decline (P <0.01) in sleep times in both male and female rats neonatally exposed to phenobarbital.
3. Neonatal phenobarbital imprints overinduction of constitutive (CYP2C6, CYP2C7, and CYP3A2) and inducible (CYP2B1, CYP2B2, and CYP3A1) isoforms of P450 in senescent rats rechallenged with nominal doses of the barbiturate
Although it is possible that a lifetime of overexpressed CYP2C6 and CYP2C7 results in the continuous, above-normal accumulation of reactive intermediary metabolites promoting tumorigenesis and a shortened life expectancy, we also considered the contribution of inducible isoforms of P450 known to be implicated in drug- and environmentally induced cancers. Accordingly, we challenged the senescent animals with a subtherapeutic dose of phenobarbital (1 mg/kg BW) that equaled 1% of its optimal inductive dose, evoking the insidious dangers to human health of comparable low levels of harmful environmental compounds. Whereas this nominal dose of phenobarbital stimulated a minimal induction of CYP2B1 and CYP2B2 (the most responsive phenobarbital inducible isoforms) in the control rats, the inductive response was several-fold greater in the 2-year-olds of both sexes neonatally treated with the barbiturate. The responsiveness of inducible CYP3A1 and constituent CYP3A2 was greater (P <0.01) in the senescent, phenobarbital-imprinted rats (Fig. 1
). Induction of CYP2C6 in females and CYP2C7 in both sexes was 40% and 30% greater, respectively, in phenobarbital-challenged, aged rats neonatally exposed to the barbiturate. This overinduction of individual P450 isoforms was reflected in the above-normal activities of the drug metabolizing enzyme system as measured by cytochrome b5, total P450, and hexobarbital hydroxylase.
|
CONCLUSIONS AND SIGNIFICANCE
While it has long been known that prepartum exposure to certain drugs and environmental chemicals can induce devastating morphologic anomalies apparent at birth, we have proposed that perinatal exposure to seemingly innocuous therapeutic levels of drugs could be the origin of subtle but permanent biochemical defects having delayed long-term health consequences. We treated neonates with therapeutic-like doses of phenobarbital for the first week of life. Twice daily observations revealed no evidence of morphologic, functional, or behavioral abnormalities throughout the greater part of the animals’ lives. General measures of well-being determined by body weight gain, linear growth, and obesity were normal. At the time of death, gross examination of the major organs (e.g., liver, kidneys, lungs, heart, gastrointestinal tract) were for the most part similar to that found in age- and sex-matched controls. In spite of this apparent normalcy, a statistically larger number (P <0.01) of barbiturate-exposed rats of both sexes died at a younger age than the controls, resulting in an 20% reduced mean life expectancy. At the time of death, necropsy results demonstrated a 2- to 3-fold increase in the incidence of tumors in barbiturate-exposed rats compared with same-sex controls.
Even though there is a growing awareness that some adult diseases may have a perinatal origin, identifying an environmental pollutant or drug ingested for a brief time during pregnancy or lactation as the cause of a disease expressed in adulthood is difficult. This inability to perceive a link between cause and effect explains why it was suppositional to identify in utero exposure to diethylstilbestrol as the cause of reproductive cancers in exposed adult offspring.
Since it is known that neonatal administration of phenobarbital induces a delayed but irreversible elevation in the rate of drug metabolism and that an individual’s hepatic P450 profile can effect their health and longevity, we have proposed that the observed adverse health consequences of perinatal exposure to the barbiturate may result, at least in part, from a permanent overexpression of individual isoforms of hepatic P450 (Fig. 2
). The results suggest that exposure of newborns to even therapeutic-like doses of phenobarbital can program the liver to continuously overexpress constitutive P450s, and when challenged by a lifetime of drug consumption and environmental insults, the liver overexpresses inducible isoforms. These factors contribute to the continuous, above-normal accumulation of reactive metabolites responsible for promoting the long-term, virtual "disconnect" effect of increasing tumorigenesis and reduced life expectancy. Coincidentally, phenobarbital is a known carcinogen that can increase the metabolism of innocuous compounds into carcinogenic and toxic metabolites. Perinatal exposure to the barbiturate has been reported to subsequently increase the risk of cancer in adult rats and children by possibly altering DNA methylation sites. Whether our results are applicable to the tens of millions of people perinatally exposed to the barbiturate is uncertain, but a published survey of the offspring of mothers administered phenobarbital during pregnancy reported that the treatment was associated with a 2.5-times higher number of deaths by early adulthood.
|
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2550fje;
This article has been cited by other articles:
|
|
 |
|
  R. Balansky, G. Ganchev, M. Iltcheva, V. E. Steele, F. D'Agostini, and S. De Flora Potent carcinogenicity of cigarette smoke in mice exposed early in life Carcinogenesis, October 1, 2007; 28(10): 2236 - 2243. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. J. Langmade, S. E. Gale, A. Frolov, I. Mohri, K. Suzuki, S. H. Mellon, S. U. Walkley, D. F. Covey, J. E. Schaffer, and D. S. Ory Pregnane X receptor (PXR) activation: A mechanism for neuroprotection in a mouse model of Niemann-Pick C disease PNAS, September 12, 2006; 103(37): 13807 - 13812. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
