title: Tryptophan deficiency arrests chromatin breakdown in secondary lens fibers of rats.
abstract: Tryptophan deficiency is known for long time to cause cataract in rats. However, up till now the underlying mechanism is still enigmatic. Histological studies showed an extended lens bow suggesting that the normal breakdown of nuclei in the lens fibres is arrested under these conditions. Using advanced ultrastructural techniques we aimed to clarify this aberrant final differentiation of lens fibres. Albino and pigmented rats were permanently or intermittently raised on a tryptophan deficient diet for 12 and 16 weeks, respectively. Rats of the same age raised on a normal diet served as controls. Lenses were treated for light and electron microscopy. For histology sections were stained for DNA and gamma-crystallins. In addition to routine transmission electron microscopy (TEM), ultrathin sections were subjected to electron tomography and energy dispersive X-ray microanalysis (EDX). Histology verified the extended lens bow for albino and pigmented rats and showed that in the intermittent period of normal diet the fibre nuclei are broken down as in controls. It was further shown that gamma-crystallins are co-localized with DNA in the nuclear domain. TEM revealed that during final differentiation nuclear chromatin becomes highly compacted in a chromosome-like manner and than rapidly evanesces in control rats. This compacted stage persists indefinitely in the tryptophan deficient rats. Electron tomography showed that during differentiation chromatin is first uncoiled to 30 nm solenoids, subsequently to highly compacted 10 nm beads-on-a-string fibrils and than is segregated from the nuclear proteins. EDX revealed that the late stage persisting nuclei consist of domains rich in DNA associated with histones and in domains with mainly proteins. This study corroborates previous findings on the final breakdown of nuclei of lens fibres. It further shows that the chromatin is ultimately uncoiled to beads-on-a-string fibrils and that as the last step chromatin is broken down at this unmasked stage. Except for this last step nuclear breakdown is identical in control and tryptophan deficient rats suggesting that it is not the availability of tryptophan for protein synthesis in general which causes the arrest. Two alternatives for this final arrest are discussed. A low tryptophan content, most pronounced in deeper cortical layers, may inhibit the late synthesis of the DNases and proteases necessary for chromatin breakdown. The radical scavenging by indoleamine 2,3-dioxygenase, which cleaves the pyrrole ring of tryptophan to form formylkynurenine using free oxygen radicals, is impaired by low levels of tryptophan. This decreased scavenging of oxygen radicals will expose the catalytic enzymes for chromatin breakdown, residing in the nucleus in an inactive form for quite a long period, to high levels of oxygen radicals and may affect the activity of these enzymes and therefore the execution of the chromatin breakdown.
authors.affiliation: Department of Ophthalmology, Leiden University Medical Centre, Leiden, The Netherlands. dr-gfjm-vrensen@hetnet.nl
authors.forename: Gijs F J M
authors.initials: GF
authors.lastname: Vrensen
authors.forename: Jan
authors.initials: J
authors.lastname: van Marle
authors.forename: Remmet
authors.initials: R
authors.lastname: Jonges
authors.forename: Wim
authors.initials: W
authors.lastname: Voorhout
authors.forename: Winrich
authors.initials: W
authors.lastname: Breipohl
authors.forename: Alfred R
authors.initials: AR
authors.lastname: Wegener
mesh_terms: D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism / Q000648:ultrastructure; D002843:Chromatin / Q000378:metabolism*; D004032:Diet; D004577:Electron Probe Microanalysis; D005260:Female; D007908:Lens, Crystalline / Q000378:metabolism* / Q000648:ultrastructure; D008854:Microscopy, Electron; D051381:Rats; D011914:Rats, Inbred BN; D017207:Rats, Sprague-Dawley; D014364:Tryptophan / Q000172:deficiency*; D038222:gamma-Crystallins / Q000378:metabolism
chemical_list: D002843:Chromatin; D038222:gamma-Crystallins; D014364:Tryptophan